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Bioengineering coagulation factor Xa substrate specificity into Streptomyces griseus trypsin Page, Michael J. 2004

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Bioengineering Coagulation Factor Xa Substrate Specificity into Streptomyces griseus Trypsin by > Michael J. Page B.Sc, Carleton University, 1998 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Biochemistry and Molecular Biology We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April 2004 © Michael J. Page, 2004  Abstract Extended substrate specificity is exhibited by a number of highly evolved members of the SI peptidase family, such as the vertebrate blood coagulation proteases. Dissection of this substrate specificity has been hindered by the complexity and physiological requirements of these proteases. In order to understand the mechanisms of extended substrate specificity, a bacterial trypsin-like enzyme, Streptomyces griseus trypsin (SGT), was chosen as a scaffold for the introduction of extended substrate specificity through structure-based genetic engineering. Recombinant and mutant SGT proteases were produced in a B.  subtilis expression  system, which constitutively secretes active protease into the extracellular medium at greater than 15 mg/L of culture. Comparison of the recombinant wild-type protease to the natively produced enzyme demonstrated near identity in enzymatic and structural properties. To begin construction of a high specificity protease, four mutants in the S1 substrate binding pocket (T190A, T190S, T190V, and T190P) were produced and examined for differences in the Arg:Lys preference. Only the T190P mutant of SGT demonstrated a significant increase in PI arginine to lysine preference - a three-fold improvement to 16:1 - with only a minor reduction in catalytic activity (kc reduction of 25%). The 1.9 A resolution crystal structure of at  T190P mutant of SGT in complex with the small molecule inhibitor benzamidine was subsequently determined. The model shows that the increased preference for Arg over Lys side chains in the SI pocket is the result of the second shell residues of the SI pocket, particularly by the N-terminal residue of the protease which does not conflict with the introduced proline ring.  ii  Using the T190P mutant of SGT as a starting point, coagulation factor Xa (FXa) substrate specificity determinants were then introduced by additional site-directed mutagenesis. To aid in purification of the recombinant proteases a hexa-histidine tag was added to the C-terminus of the protein. Addition of the purification tag reduced the ability of the expression host to produce the enzyme (3 mg/L of culture) but simplified the purification of SGT from the culture medium. Various combinations of a two-residue loop and a number of point mutations at positions 99, 172, 174, 180, and 217 were constructed and characterized in SGT. The mutant bearing mutations at all positions except residue 217 demonstrated a moderate preference for FXa substrates as determined using chromogenic synthetic peptides. However, the kinetic properties of the mutant enzyme suggested that the 172-loop, a member of the S3/S4 substrate binding pocket, is not in a conformation similar to FXa. Addition of the Y217E mutation was designed to stabilize the loop but led to a specific protease similar to coagulation factor XIa and not FXa. These results confirm the evolutionary relationship amongst the vertebrate coagulation proteases and demonstrate the importance and flexibility of the 172-loop. Further, Na binding, a novel property found in several coagulation +  proteases, is suggested to play a role in stabilization of the 172-loop and in turn played an important role in the evolution of the vertebrate coagulation cascade.  iii  Table of Contents  Abstract  ii  Table of Contents  iv  List of Tables  vii  List of Figures  viii  List of Abbreviations  x  Acknowledgments  xi  Chapter 1. Introduction  1  1.1 Focus of Research  1  1.2 Overview  1  1.3 Nomenclature of Proteases  4  1.4 Definition of Substrate Specificity  6  1.5 Aspartic, Cysteine, Metallo-, and Threonine Proteases  7  1.6 Catalytic Mechanism of Serine Proteases  9  1.7 Blood Coagulation and Fibrinolysis  14  1.8 Substrate Specificity of Serine Proteases  17  1.9 Genetic Manipulation of Serine Proteases  20  1.10 Streptomyces griseus Trypsin  21  1.11 Statement of Hypothesis  22  1.12 Objectives and Outline  22  Chapter 2. Recombinant Protein Expression of Streptomyces griseus Trypsin in Bacillus subtilis 25  2.1 Introduction  25  2.2 Materials & Methods 2.2.1 Plasmids, Bacterial Strains, and Growth Conditions 2.2.2 DNA manipulation 2.2.3 Protein Purification 2.2.4 Kinetic Analysis 2.2.5 Crystallization  28 28 28 29 30 30  2.3 Results and Discussion  ,  31  iv  2.3.1 2.3.2 2.3.3 2.3.4 2.3.5  Production and Purification of Recombinant SGT Kinetic Analysis Crystallization and Structure Determination Comparison of the recombinant and Pronase-derived crystal structure Wild-type Native and Recombinant SGT Substrate Binding  2.4 Conclusions  31 36 37 40 41 42  Chapter 3. Engineering the Primary Substrate Specificity of Streptomyces griseus Trypsin  43  3.1 Introduction  43  3.2 Materials & Methods 3.2.1 DNA Manipulation and Protein Purification 3.2.2 Kinetic Analysis 3.2.3 Crystallization and Structure Determination  44 44 45 45  3.3 Results and Discussion 3.3.1 Production of SGT Mutants 3.3.2 Kinetic Analysis 3.3.3. Crystallization and Structure Refinement 3.3.4 Ca* binding site of B. subtilis derived SGT 3.3.5 T190S and the Loss of y-CH 3.3.6 T190V and the Effect of a Branched Side Chain 3.3.7 T190A and the Loss of y-OH 3.3.8 T190P 3.3.9 Second Shell Residues  46 46 46 48 50 51 53 53 54 58  3.4 Conclusions  58  +  3  Chapter 4. Engineering Coagulation Factor Xa Substrate Specificity into Streptomyces griseus Trypsin 59  4.1 Introduction 4.1.1 Overview 4.1.2 Choice of Mutations to Mimic FXa-like Specificity  59 59 62  4.2 Materials & Methods 4.2.1 Plasmids, Bacterial Strains, and Growth Conditions 4.2.2 Construction of a Hexahistidine-tagged SGT 4.2.3 Sequence analysis of the SI Family peptidases 4.2.4 DNA Manipulation 4.2.5 Purification of His-tagged SGT and Mutants thereof 4.2.6 Characterization of Substrate Specificity 4.2.7 Macromolecular Substrate Specificity  65 65 65 67 67 68 69 69  4.3 Results & Discussion 4.3.1 Production of His-tagged SGT 4.3.2 Techniques for Characterization of Substrate Specificity of Serine Proteases  70 70 71  v  4.3.3 4.3.4 4.3.5 4.3.6 4.3.7 4.3.8  Extended Substrate Specificity of SGT Effect of the 99-loop on the Substrate Specificity of SGT Mechanisms of P3 Selectivity in SI Family Peptidases Role of the 172-loop and Residue 217 in the SI Peptidases Additional Elements Needed for Reconstructing FXa-like Specificity Utility of a FXa-like Protease  4.4 Conclusions & Future Directions  72 74 76 79 86 89 92  Chapter 5. General Discussion and Outlook  94  5.1 Substrate specificity determinants of the SI family of Serine Proteases  94  5.2 Molecular Evolution of the SI family of Serine Proteases  98  5.3 Paper, Rock, Scissors Genetic Screening of Trypsin-like Proteases  99  5.5 Future Opportunities  107  5.6 Significance of the Work  108  5.7 Conclusions  109  Appendix A: Structural Alignment of Selected SI Family Peptidases  110  Bibliography  Ill  vi  List of Tables Table 1.1 A short list of biological processes involving proteolysis  3  Table 1.2 A short list of pathologies involving abnormal proteolysis  3  Table 1.3 Select examples of successful protein engineering of serine proteases Table 2.1 Purification table for recombinant SGT (bSGT) from B.  21  subtilis extracellular  supernatant  34  Table 2.2 PI arginine to lysine preference of SGT enzymes  37  Table 2.3 Data collection and refinement statistics of wild-type recombinant SGT  39  Table 3.1 Oligonucleotides used to mutate residue 190 in the SGT gene  45  Table 3.2 ES-MS analysis of the four mutants of SGT  46  Table 3.3 PI Arginine to Lysine preference of mutant SGT enzymes  47  Table 3.4 Ki values of benzamidine for recombinant SGT and the four mutant forms  48  Table 3.5 Data collection and refinement statistics for the T190P mutant of SGT  49  Table 4.1 Mutants of SGT constructed to mimic the substrate specificity of FXa  62  Table 4.2 Oligonucleotides used to mutate the SGT gene to mimic residues found in FXa....68 Table 4.3 Steady-state kinetic parameters for the hydrolysis of a series of p-nitroanilide chromogenic substrates by the YSFMP mutant of SGT  83  Table 4.4 Steady-state kinetic parameters for the hydrolysis of a series of p-nitroanilide chromogenic substrates by the YSFMPE mutant of SGT  85  Table 4.5 Cleavage sites of proteases used in processing recombinant proteins  90  Table 5.1 Substrate specificities of SI family peptidases  96  Table 5.2 Substrate specificity determinants of SI family sub-family A peptidases  100  Table 5.3 Inhibition constants of ecotin against a variety of SI family peptidases  104  vii  List of Figures  Figure 1.1 Distribution of identified proteases based on catalytic type  2  Figure 1.2 Derivation of the Michaelis-Menten equation  6  Figure 1.3. Catalytic mechanism of a typical zinc metalloprotease  9  Figure 1.4 Catalytic triad of serine proteases  10  Figure 1.5 Three dimensional structure of a typical serine protease  12  Figure 1.6 Catalytic mechanism of a serine protease  13  Figure 1.7 Overview of vertebrate blood coagulation  15  Figure 1.8 Protein domains of the vertebrate blood coagulation proteases  16  Figure 1.9 Schecter & Berger nomenclature of protease specificity  18  Figure 2.1 Plasmid map of SGT gene cloned into pWB980  33  Figure 2.2. ES-MS spectrum of purified recombinant SGT  35  Figure 2.3 SDS-PAGE of purified recombinant SGT  36  Figure 2.4 Ramachandran plot of the crystal structure of recombinant wild-type SGT  38  Figure 2.5 Superimposition of the C traces of native and recombinant wild-type SGT  41  Figure 3.1 Ramachandran plot of the crystal structure of the T190P mutant of SGT  50  Figure 3.2 Comparison of the Ca binding site in SGT enzymes  52  Figure 3.3 Comparison of the SI binding pocket in SGT enzymes  56  a  2+  Figure 4.1 Residues involved in the extended substrate specificity of coagulation proteases .63 Figure 4.2 Plasmid construction for the production of recombinant His-tagged SGT  66  Figure 4.3 Purification of a typical His-tagged mutant of SGT  71  Figure 4.4 Substrates used to characterize mutants of SGT with altered substrate specificity 73 Figure 4.5 Normalized kcat/K values for the T190P, LP and YP mutants of SGT  75  Figure 4.6 S3 & S4 binding pockets of FXa  77  Figure 4.7 S3 and S4 binding pockets of FVIIa  78  Figure 4.8 Conformation of the 172-loop in SI peptidases  80  m  Figure 4.9 Normalized KJKm values for the YFP and YSFP mutants of SGT  82  Figure 4.10 Na -binding site in thrombin  88  Figure 4.11 Prothrombin processing by mutants of SGT  91  +  viii  Figure 5.1 Simplified phylogenetic tree of the SI family of peptidases  95  Figure 5.2 Theory behind the Paper, Rocks, Scissors genetic screen  103  Figure 5.3 Dimeric structure of ecotin  104  Figure 5.4 Structure of GFP  106  ix  List of Abbreviations  A  Angstrom unit (1 A = 0.1 run)  AMC  Aminomethylcoumarin  aPC  Activated protein C  B  Crystallographic thermal factor (A )  Bz  Benzoyl  dNTP  Dinucleotidetriphosphate  Fo, Fc  Observed and calculated structure factors  FX  Coagulation factor X (similar for other coagulation proteases)  FXa  Activated coagulation factor X (similar for other coagulation proteases)  kcat  Catalytic constant  K  Michaelis constant  2  m  LB  Luria - Bertani broth  NMWL  Nominal molecular weight limit  PCR  Polymerase Chain Reaction  Pip  Pipecoyl  pNA  para-nitroanilide  r.m.s.  Root mean squared  Rfree  R factors based test set of excluded reflections  Rmerge  Shk! Si |li(hkl) - <I>(hkl)(| / Shk! Si Ii(hkl)  R-cryst  £ ||F bs| - |F lc|| / 2 |F bs|  S.D.  Standard deviation at 95% confidence interval  0  ca  0  SDS-PAGE Sodium dodecyl sulfate - polyacrylamide gel electrophoresis Tos  Tosyl  Tris-HCI  Tris(hydroxymethyl)aminomethane hydrochloride  Acknowledgments I a m indebted to R o s s and J e f f for countless hours o f g o o d times, their use o f " N u m b e r 1", and their a b i l i t y to define n e w verbs. I w o u l d e s p e c i a l l y l i k e to thank S u i - L a m W o n g for p r o v i d i n g the essential c o m p o n e n t s o f the e x p r e s s i o n system. I appreciate m y parents f o r their constant support b o t h e m o t i o n a l l y and f i n a n c i a l l y throughout m y life. I thank m y friends, for their h e l p , a d v i c e , time and patience. I e s p e c i a l l y thank I a i n , M a r t y , I s m a i l , A n g u s , M i k e K . , and M a r k and w i s h t h e m the best i n a l l endeavors. A s p e c i a l thanks to T a n y a for a l l o f her h e l p f u l c o m m e n t s .  T h i s thesis is dedicated to m y b e l o v e d D o e  xi  Chapter 1. Introduction 1.1 Focus of Research How do you create a highly efficient and highly specific enzyme? Over several million years, nature has evolved many enzymes that possess high degrees of specificities. Using methods of genetic manipulation, properties of enzymes can be altered, including their substrate specificity. The present dissertation involves taking a primitive bacterial enzyme and adding substrate specificity where little existed previously. By doing so, we will learn about how molecular evolution has produced substrate specificity in one family of enzymes, the serine proteases. I will use a human protease involved in blood coagulation as our guide and then extend the concepts learned to a system that could produce a variety of other specificities. To begin, I will discuss what is a protease, what do they do and why they are important. From there, I will describe the system and the methods applied to generate a specific protease, what has been done by others and then by the author. Lastly, I will discuss the opportunities that may result from this work.  1.2 Overview Proteolytic enzymes play a diverse number of roles in a variety of essential biological processes, both as non-specific catalysts of protein degradation and as highly specific agents that control physiological events. Hydrolysis of a peptide bond is the key function that proteases fulfill in vivo, and this may result in the activation or destruction of its substrate. Numerous biological processes involving proteolytic activity have been characterized and a wealth of information has been gathered on the five major catalytic classes of these enzymes (Figure 1.1, Table 1.1). Roughly 2 % of all genes in most organisms are proteases, second  1  o n l y to transcription factors. H e n c e , the importance o f these types o f e n z y m e s i n b i o l o g i c a l and c o m m e r c i a l settings c a n n o t be understated. Unknown  Figure 1.1 D i s t r i b u t i o n  o f identified proteases based o n c a t a l y t i c type.  N u m e r o u s p a t h o l o g i c a l c o n d i t i o n s are the result o f e x c e s s i v e o r i n s u f f i c i e n t proteolytic a c t i v i t y ( T a b l e 1.2). T h e s e c l i n i c a l situations c a n arise f r o m genetic defects i n the proteases themselves, their natural substrates o r their natural i n h i b i t o r s . A n u m b e r o f a c a d e m i c laboratories a n d p h a r m a c e u t i c a l c o m p a n i e s are d e v o t e d to the p r o d u c t i o n o f therapeutic products, s u c h as s m a l l m o l e c u l e i n h i b i t o r s o r r e c o m b i n a n t proteins, to m i n i m i z e the effects o f protease-related pathologies  [1,2]. G i v e n the large n u m b e r o f c l o s e l y related  proteases f o u n d i n m a n , the d e s i g n o f potent i n h i b i t o r s w i t h m i n i m a l cross r e a c t i v i t y is an arduous task. D e t a i l e d i n f o r m a t i o n o n the active site g e o m e t r y a n d e l e c t r o n i c c o n f i g u r a t i o n o f the protease is a requirement for proper i n h i b i t o r d e s i g n . T h e s e studies have b e e n h a m p e r e d  2  Biological Process  Proteolytic Event  Ref.  Apoptosis  Control of physiological cell death  [3]  Blood Coagulation  Proteolytic cascades of clot formation, fibrinolysis  [4]  Blood Pressure  Renin-angiotensin and kallikrein-kinin systems  [5]  Digestion Fertilization Immunity  Breakdown of protein into tri- and dipeptides; liberation of hormones promoting digestion Sperm-Egg interaction, ovulation, ovum implantation and parturition Complement activation, antigen presentation, chemokine and chemotaxin activation;  Intracellular Proteasome-Ubiquitin system protein level Protein Zymogen activation and protein sorting Processing Tissue Turnover and repair of the extracellular environment Remodeling Table 1.1 A short list of biological processes involving proteolysis.  [6] [7] [8] [9] [10] [11]  Pathology  Proteolytic Event  Ref.  Alzheimer's Disease  Processing of amyloid precursor protein  [12]  Cancer  Regulation of apoptosis, tumor growth and invasion  [3]  Chronic Inflammation  Excessive activation of pro-inflammatory cytokines  [13]  Hemophilia  Insufficient levels of coagulation factor activity and slowed clot formation  [14]  Myocardial Infarction  Unregulated coagulation leading to restricted blood flow  [15]  Parasite Infection  Regulation of the parasitic life-cycle  [16]  Processing of viral coat proteins and other essential replication machinery required for viral infection Table 1.2 A short list of pathologies involving abnormal proteolysis.  Viral Replication  3  [17]  b y the a b i l i t y to p r o d u c e large quantities o f the target e n z y m e and detailed three d i m e n s i o n a l structural m o d e l s o f them. Industrial usage o f p r o t e o l y t i c e n z y m e s is w i d e s p r e a d and c o m m e r c i a l l y important. F o r e x a m p l e , the p r o d u c t i o n o f the protease s u b t i l i s i n for use i n detergents is o n the scale o f tons per a n n u m and accounts for 4 0 % o f e n z y m e sales w o r l d w i d e . O t h e r i n d u s t r i a l a p p l i c a t i o n s o f proteases i n c l u d e the p r o d u c t i o n o f f o o d stuffs, leather, p h a r m a c e u t i c a l s , diagnostic reagents, waste management, and silver r e c o v e r y [18,19]. R e c e n t advances i n m o l e c u l a r b i o l o g y t e c h n o l o g y have a l l o w e d for the d e v e l o p m e n t o f proteases w i t h i m p r o v e d properties for i n d u s t r i a l use. S o m e successful e x a m p l e s o f p r o t e i n e n g i n e e r i n g i n c l u d e thermostability, resistance to o x i d a t i o n , alteration o f p H o p t i m a , and increased catalytic e f f i c i e n c y [20]. H o w e v e r , the use o f h i g h l y s p e c i f i c proteases for site s p e c i f i c p r o t e o l y s i s has for the m o s t part been l i m i t e d to a c a d e m i c endeavors due to the difficulties associated w i t h p r o d u c i n g h i g h p u r i t y e n z y m e s f r o m the c o m p l e x b i o l o g i c a l systems f r o m w h i c h they d e r i v e . In the present dissertation, a n o v e l system has been d e v e l o p e d to p r o d u c e a r e c o m b i n a n t protease and the substrate s p e c i f i c i t y o f the e n z y m e altered. T h e results contribute to o u r understanding o f the substrate s p e c i f i c i t y determinants o f the t r y p s i n - l i k e f a m i l y o f serine proteases. F u r t h e r m o r e , a f r a m e w o r k f o r d e s i g n i n g n o v e l s p e c i f i c i t i e s not o b s e r v e d i n nature is then presented based o n the w o r k .  1.3 Nomenclature of Proteases P r i o r to a literature r e v i e w , a f e w w o r d s must be m e n t i o n e d on the n o m e n c l a t u r e u s e d to describe p r o t e o l y t i c e n z y m e s . T h e International U n i o n o f B i o c h e m i c a l and M o l e c u l a r B i o l o g y N o m e n c l a t u r e C o m m i t t e e ( I U B M B - N C ) denotes hydrolases acting o n peptide b o n d s  4  as E . C . 3 . 4 , yet o n l y a s m a l l fraction o f the k n o w n proteases have b e e n g i v e n f u l l c l a s s i f i c a t i o n . G i v e n the w i d e d i s t r i b u t i o n o f p r o t e o l y t i c e n z y m e s and their h i s t o r i c a l s i g n i f i c a n c e , a variety o f c o m m o n names are used. P a p a i n , c o m m o n l y u s e d to tenderize meat, o w e s its name to the p a p a y a plant f r o m w h i c h it is d e r i v e d . O t h e r proteases are n a m e d based o n the p h y s i o l o g i c a l process i n w h i c h they were first d e s c r i b e d , such as c o a g u l a t i o n factor X w h i c h is i n v o l v e d i n vertebrate b l o o d c o a g u l a t i o n . U n f o r t u n a t e l y , a s i g n i f i c a n t n u m b e r o f proteases o w e their name to h a v i n g s i m i l a r b i o c h e m i c a l properties yet are i n v o l v e d i n disparate b i o l o g i c a l processes or h a v i n g different catalytic m e c h a n i s m s . F o r e x a m p l e , t w o o f the m a n y proteases f o u n d i n the h u m a n l i v e r , cathepsin B and cathepsin D , are n a m e d s i m i l a r l y but b e l o n g to the cysteine and aspartic protease f a m i l y , r e s p e c t i v e l y . T h u s , the n o m e n c l a t u r e for each particular protease has b e c o m e rather m u d d l e d w i t h c o n v e n t i o n . A variety o f terms have been u s e d to refer to e n z y m e s that c a t a l y z e the h y d r o l y s i s o f a peptide b o n d . T h e term peptidase has been suggested b y the I U B M B - N C as a preferred alternative to protease [21]. Peptidase refers m o r e e a s i l y to b o t h exopeptidases (enzymes that c l e a v e peptide bonds at the ends o f a p o l y p e p t i d e c h a i n ) , endopeptidases (those that c l e a v e peptide bonds i n the m i d d l e o f p o l y p e p t i d e c h a i n s ) , and oligopeptidases (those that c l e a v e o n l y short p o l y p e p t i d e s ) . E x o p e p t i d a s e s are further s u b d i v i d e d into aminopeptidases ( c l e a v i n g at the N - t e r m i n u s ) and carboxypeptidases ( c l e a v i n g at the C - t e r m i n u s ) . O t h e r terms f o u n d i n the literature i n c l u d e proteinase and p r o t e o l y t i c e n z y m e . I n the present thesis, the terms protease and peptidase w i l l be used interchangeably to describe the e n z y m e s studied i n the research; a l l o f w h i c h are endopeptidases.  5  1.4 Definition of Substrate Specificity E n z y m e k i n e t i c s are t r a d i t i o n a l l y d e f i n e d b y M i c h a e l i s - M e n t e n k i n e t i c s w h i c h a p p l y a steady state a s s u m p t i o n to the r e a c t i o n ( F i g u r e 1.2). Substrate s p e c i f i c i t y is t r a d i t i o n a l l y defined b y the ratio o f the m a x i m u m reaction v e l o c i t y c a t a l y z e d b y the e n z y m e per u n i t t i m e (kcat) to the M i c h a e l i s - M e n t e n constant ( K ) . kc is e q u a l to the m a x i m a l r e a c t i o n v e l o c i t y m  al  (Vmax) d i v i d e d b y the total amount o f e n z y m e i n the reaction. In theory, b o t h constants (kcat and K ) are l i n k e d m a t h e m a t i c a l l y as K m  (K  m  m  is dependent o n the rates o f each step i n the reaction  = (k.i + k 2 ) / ki). I n the present study, the traditional d e f i n i t i o n o f substrate s p e c i f i c i t y  w i l l be a p p l i e d (S = k ^ / K m ) .  B E  +s  ES  —> E + p  [ES] = k, [E][S] / (k, + k )  (1)  Fraction of enzyme in ES: F = (ES] / ([E] + [ES])  (2)  (1) + (2) yields: F= [S] / {((k + k ) / k, ) + [S]} 2  (3)  Initial rate of reaction: v = v  F  (4)  2  Initial Rate of Reaction: v = d[P] / dt = k [ES]  1  Steady state assumption: d[ES] / dt = 0  0  Rate of ES formation = Rate of ES breakdown  (3) + (4) yields: v = v 0  k,[El[Sl = k.^ES] +k [ES]  m a x  m a x  [S] / «(k., + k ) / k,) 2  MichaelisConstant: K = ( k + k ) / k (valid when k , » k )  2  1  2  + [S]}  1  (5) (6)  2  (5)+ (6)  Figure 1.2 D e r i v a t i o n o f  v =v 0  max  [S]/(K  +[S])  the M i c h a e l i s - M e n t e n equation. F o r a t y p i c a l e n z y m e  c a t a l y z e d reaction (A) the rate o f change o f the e n z y m e ( E ) i n the enzyme-substrate c o m p l e x ( E S ) is assumed to be constant throughout the reaction. T h e M i c h a e l i s M e n t e n equation c a n then be d e r i v e d (B) b y i n t r o d u c i n g the M i c h a e l i s constant ( K ) , m  w h i c h is o n l y true i f the rate o f E S f o r m a t i o n (ki) is m u c h larger than the rate o f p r o d u c t f o r m a t i o n (k ). 2  6  1.5 Aspartic, Cysteine, Metallo-, and Threonine Proteases H i s t o r i c a l l y , four m e c h a n i s t i c classes o f p r o t e o l y t i c e n z y m e s have been r e c o g n i z e d based o n their catalytic m e c h a n i s m - aspartic, cysteine, m e t a l l o - , and serine proteases. W i t h the advent o f w h o l e genome s e q u e n c i n g this c l a s s i f i c a t i o n system has b e c o m e inadequate as the variety o f catalytic m e c h a n i s m s i d e n t i f i e d i n nature has e x p a n d e d r a p i d l y . A t present, n e a r l y 18,000 gene sequences for peptidases have been i d e n t i f i e d and over 2 0 0 0 peptidases have been characterized. Barrett has d e v i s e d a c l a s s i f i c a t i o n scheme based o n statistically significant s i m i l a r i t i e s i n sequence and structure o f a l l k n o w n p r o t e o l y t i c e n z y m e s , and terms this database M E R O P S ( w w w . m e r o p s . a c . u k ) [22,23]. T h i s s y s t e m d i v i d e s a l l k n o w n proteases into 4 0 clans and over 169 s u b - f a m i l i e s , not i n c l u d i n g a group o f putative proteases o f u n k n o w n m e c h a n i s m . O n l y the four h i s t o r i c c l a s s i f i c a t i o n s o f p r o t e o l y t i c e n z y m e s w i l l be m e n t i o n e d here. H y d r o l y s i s o f a p o l y p e p t i d e b a c k b o n e requires three k e y m e c h a n i s t i c hurdles to be o v e r c o m e for efficient catalysis to p r o c e e d . P e p t i d e bonds are p a r t i c u l a r l y stable due to the electron resonance b e t w e e n the amide n i t r o g e n and c a r b o n y l g r o u p o f the b o n d . B y the use o f a general a c i d , proteases o v e r c o m e this partial d o u b l e b o n d character t h r o u g h the generation o f a n e g a t i v e l y c h a r g e d tetrahedral intermediate that is s t a b i l i z e d b y the active site. S e c o n d l y , water is a p o o r n u c l e o p h i l e and must be activated, t y p i c a l l y v i a a general base. L a s t l y , amines are p o o r l e a v i n g groups and must be e x p e l l e d f r o m the active site p r i o r to c o m p l e t i o n o f the catalytic c y c l e [24]. Proteases a c c o m p l i s h these tasks e f f i c i e n t l y and increase the rate o f reaction ~ 1 0 - f o l d o v e r the u n c a t a l y z e d reaction. M o r e o v e r , proteases c a n catalyze s i m i l a r 1 0  reactions i n the h y d r o l y s i s o f amides, esters, a n i l i d e s , and thioesters.  7  A s p a r t i c proteases and metalloproteases catalyze the h y d r o l y s i s o f the p o l y p e p t i d e b a c k b o n e through activation o f a water m o l e c u l e . M e t a l l o p r o t e a s e s c o m p r i s e the s e c o n d largest f a m i l y o f proteases k n o w n i n nature and t y p i c a l l y u t i l i z e a z i n c i o n i n their active site; h o w e v e r , c o b a l t or manganese are also f o u n d ( F i g u r e 1.3). In m a n y metalloproteases, a s i n g l e m e t a l i o n is u t i l i z e d i n the catalysis; h o w e v e r t w o m e t a l i o n s a c t i n g c o c a t a l y t i c a l l y are t y p i c a l l y f o u n d i n proteases c o n t a i n i n g cobalt or manganese. T h r e e a m i n o a c i d side c h a i n s , t y p i c a l l y H i s , G l u , A s p or L y s residues, are i n v o l v e d i n the c o - o r d i n a t i o n o f the m e t a l i o n and at least one other residue is r e q u i r e d for catalysis [25]. T h e catalytic residue is t y p i c a l l y G l u i n m a n y metallopeptidases but alternatives exist. A c t i v a t i o n o f the water m o l e c u l e i n the aspartic f a m i l y o f proteases is the result a p a i r o f A s p residues that co-ordinate the activated water m o l e c u l e [26]. In contrast to n u c l e o p h i l i c attack o f the a m i d e b a c k b o n e b y an activated water m o l e c u l e , cysteine, serine, and threonine peptidases u t i l i z e an a m i n o a c i d side c h a i n . C y s t e i n e proteases c o m p r o m i s e the t h i r d largest f a m i l y o f k n o w n peptidases and e m p l o y a n u c l e o p h i l i c s u l f h y d r y l f r o m a cysteine residue to c a t a l y z e the h y d r o l y s i s o f an a m i d e b o n d [27]. A l t h o u g h less abundant i n nature, threonine peptidases are deeply i n v o l v e d i n the k e y b i o l o g i c a l process o f i n t r a c e l l u l a r degradation o f p o l y p e p t i d e s b y the proteasome [28]. T h e o v e r a l l catalytic m e c h a n i s m o f b o t h o f these f a m i l i e s o f protease is m o r e s i m i l a r to that f o u n d i n serine proteases, where a n u c l e o p h i l e as w e l l as a p r o t o n d o n o r is r e q u i r e d for catalysis. T h e p r o t o n d o n o r i n a l l cysteine and threonine peptidases w h i c h have b e e n i d e n t i f i e d is a H i s residue, w h i c h is also true o f a l l k n o w n serine proteases.  8  Figure 1.3. C a t a l y t i c m e c h a n i s m o f a t y p i c a l z i n c metalloprotease. T h e z i n c i o n serves to p o l a r i z e the c a r b o n y l g r o u p o f the substrate as w e l l as facilitate the deprotonation o f the water m o l e c u l e . S e v e r a l h y d r o g e n b o n d i n g partners stabilize the intermediates but are not i n c l u d e d i n the d i a g r a m f o r s i m p l i c i t y .  1.6 Catalytic Mechanism of Serine Proteases N e a r l y a third o f a l l k n o w n proteases are c l a s s i f i e d i n the serine protease f a m i l y o f e n z y m e s . T h e f a m i l y n a m e stems f r o m the n u c l e o p h i l i c serine residue i n the active site o f the e n z y m e , a n d the catalytic p o t e n c y o f this residue is dependent o n the A s p - S e r - H i s charge r e l a y s y s t e m o r c a t a l y t i c triad w h i c h w a s o r i g i n a l l y p r o p o s e d b y B l o w o v e r 3 0 years ago ( F i g u r e 1.4) [29]. T h e s e three residues are f o u n d i n an i d e n t i c a l structural p o s i t i o n i n four different t h r e e - d i m e n s i o n a l p r o t e i n folds that c a t a l y z e the h y d r o l y s i s o f peptide b o n d s ,  9  suggesting four distinct e v o l u t i o n a r y o r i g i n s . C o m m o n e x a m p l e s o f these folds are represented b y c h y m o t r y p s i n , s u b t i l i s i n , carboxypeptidase Y , and C l p protease. A n u m b e r o f other e n z y m e f a m i l i e s , i n c l u d i n g asparaginases, esterases, acylases, and P-lactamases, u t i l i z e the A s p - S e r - H i s catalytic triad or variants to generate a strong n u c l e o p h i l e and p r o m o t e catalysis [30]. F o r the r e m a i n d e r o f the i n t r o d u c t i o n , I w i l l l i m i t the d i s c u s s i o n to the c h y m o t r y p s i n f a m i l y ( S I peptidase f a m i l y ) o f serine proteases, w h i c h i n c l u d e s trypsins and elastases. M o r e o v e r , I w i l l u t i l i z e the c h y m o t r y p s i n n u m b e r i n g system suggested b y B l o w to refer to a particular a m i n o a c i d residue. It must be noted that m a n y o f the concepts discussed i n r e l a t i o n to c h y m o t r y p s i n - l i k e proteases a p p l y s i m i l a r l y to other types o f proteases.  His57 /  Asp 102  His57 /  Ser195  Asp102 — C H ,  CHJ  Figure 1.4  Ser195  C a t a l y t i c triad o f serine proteases. T h e A s p , H i s , and Ser c o m b i n a t i o n is  f o u n d i n serine proteases and other e n z y m e f a m i l i e s that require a n u c l e o p h i l i c serine side c h a i n .  C e n t r a l to the catalytic triad i s the existence o f a h y d r o g e n b o n d between residue A s p l 0 2 and H i s 5 7 , w h i c h facilitates the abstraction o f the p r o t o n f r o m S e r l 9 5 and generates a potent n u c l e o p h i l e . S o m e c o n t r o v e r s y exists o v e r whether this h y d r o g e n b o n d c a n be described as a l o w barrier h y d r o g e n b o n d ( L B H B ) , an instance where the p K values between  10  the d o n o r and acceptor are matched. R e j e c t i o n o f the L B H B theory m a i n l y stems f r o m the argument that it w o u l d p r o v i d e no s i g n i f i c a n t i m p r o v e m e n t to catalytic rate enhancement [31,32]. Increasing e x p e r i m e n t a l and theoretical data are supporting this theory and the debate continues [33,34]. S t a b i l i z a t i o n o f the catalytic triad is mediated t h r o u g h a n e t w o r k o f a d d i t i o n a l h y d r o g e n bonds p r o v i d e d b y several h i g h l y c o n s e r v e d a m i n o a c i d residues s u r r o u n d i n g the triad, p a r t i c u l a r l y A l a 5 6 and S e r 2 1 4 i n the c h y m o t r y p s i n f a m i l y o f serine proteases. S i g n i f i c a n t effort has been p l a c e d i n the d e v e l o p m e n t o f s m a l l m o l e c u l e c o m p o u n d s that m i m i c the a c t i v i t y the catalytic triad, but have met w i t h l i m i t e d success due to the c o m p l e x i t y o f the c h e m i s t r y i n v o l v e d to generate the n u c l e o p h i l i c serine. A c t i v a t i o n o f c h y m o t r y p s i n - l i k e serine proteases requires p r o t e o l y t i c p r o c e s s i n g o f a n i n a c t i v e z y m o g e n precursor p r o t e i n . T h i s cleavage occurs at the i d e n t i c a l p o s i t i o n i n a l l k n o w n members o f the f a m i l y : between residues 15 and 16 [24]. T h e n e w l y created N terminus produces a c o n f o r m a t i o n a l change i n the e n z y m e and stabilizes the o x y a n i o n h o l e and substrate b i n d i n g site t h r o u g h f o r m a t i o n o f an electrostatic interaction w i t h A s p l 9 4 [29]. T w o P-barrel d o m a i n s , each f o r m e d b y six anti-parallel p-strands, and a C - t e r m i n a l a - h e l i x c o m p r i s e the mature f o r m o f the e n z y m e ( F i g u r e 1.5). B o t h the catalytic residues and substrate b i n d i n g site l i e i n the cleft between the P-barrel d o m a i n s , and enzyme-substrate interactions o c c u r w i t h b o t h d o m a i n s . A m i n i m u m o f three d i s u l p h i d e bonds is r e q u i r e d to stabilize the o v e r a l l structure; h o w e v e r f i v e or six are c o m m o n l y f o u n d i n the f a m i l y o f enzymes.  11  Figure 1.5 T h r e e d i m e n s i o n a l structure o f a t y p i c a l serine protease o f the S I f a m i l y o f peptidases. C o m p o n e n t s o f the catalytic triad are s h o w n i n stick f o r m a n d are l o c a t e d i n between the t w o (3-barrel d o m a i n s .  F i g u r e 1.6 depicts the g e n e r a l l y accepted m e c h a n i s m o f serine protease c a t a l y z e d h y d r o l y s i s o f a peptide b o n d [24]. I n i t i a l l y , the h y d r o x y l o x y g e n o f S e r 195 attacks the c a r b o n y l o f the peptide substrate as a result o f H i s 5 7 i n the catalytic t r i a d a c t i n g as a g e n e r a l base (Steps I and II). T h e o x y a n i o n tetrahedral intermediate is s t a b i l i z e d b y the b a c k b o n e atoms o f G l y l 9 3 (not depicted) and S e r l 9 5 that generate a p o s i t i v e l y c h a r g e d p o c k e t w i t h i n the active site (Step III). C o l l a p s e o f the tetrahedral intermediate generates the a c y l - e n y z m e intermediate a n d s t a b i l i z a t i o n o f the n e w l y created N - t e r m i n u s is m e d i a t e d b y H i s 5 7 (Steps I V and V ) . E v i d e n c e f o r the existence o f the a c y l - e n z y m e intermediate w a s p r o v i d e d i n 1954 b y H a r t l e y a n d K i l b e y [35]. In these i n i t i a l experiments a pre-steady state burst o f p r o d u c t  12  His 57  His57  /  /  Ser195  Ser195  H,C  \  \  CH,  /  /  \  a---"  o.  /" N — C  Asp102  CH.  H  CH,  H  /  ^ O  O  Asp102—CH,  Acyl-enzyme Intermediate His57  II  His57  VI  /  V\ \  „J  Ser195  H,C  \ w  7  °. Asp102  a-  Ser19S  O  N—C'l  /  / CM;  V  H  Asp102  O  C,  CH,  Formation of the Michaelis Complex His57  111  His57  VII  / H,C  /  Ser195  2  \CH,  Asp102  CH,  rr.  a-  CHj  a--' / Asp102 C H  V_e  ;  Tetrahcdral Intermediate  Tetrahcdral Intermediate  His57  IV  \  . © _  / o.  Sen 95  HC  His57  VIII  /  Serf 95  H,C  \  /  Serf 95  \  CHJ  CH,  ,  / H  O  ' * - H - O '  a--' H  o—c  BO  Asp102  Asp102—CH,  CH,  H  O  Free C-terminus generated  Figure 1.6 C a t a l y t i c  m e c h a n i s m o f a serine protease. F o r m a t i o n o f a tetrahedral  intermediate is the k e y c o n f o r m a t i o n a l step i n the a c y l a t i o n a n d d e a c y l a t i o n reactions.  13  c o r r e c t l y i d e n t i f i e d that a b o n d to a h y d r o x y l m o i e t y w i t h i n c h y m o t r y p s i n was i n v o l v e d i n the reaction m e c h a n i s m . I n the s e c o n d h a l f o f the m e c h a n i s m , a water m o l e c u l e displaces the free p o l y p e p t i d e fragment and attacks the a c y l - e n z y m e intermediate (Step V I ) . A g a i n , the o x y a n i o n h o l e stabilizes the s e c o n d tetrahedral intermediate o f the p a t h w a y and c o l l a p s e o f this intermediate liberates a n e w C - t e r m i n u s .  1.7 Blood Coagulation and Fibrinolysis Vertebrate b l o o d c o a g u l a t i o n and f i b r i n o l y s i s c a n serve as a useful p a r a d i g m for the study o f p r o t e o l y s i s i n a b i o l o g i c a l setting. T h e process serves as a m o d e l for pathologies associated w i t h i m p r o p e r p r o t e o l y s i s , m o l e c u l a r e v o l u t i o n through gene d u p l i c a t i o n and d i v e r g e n c e , as w e l l as understanding m o l e c u l a r r e c o g n i t i o n a n d substrate s p e c i f i c i t y . I n i t i a l l y r e c o g n i z e d as a cascade o f events that leads to a m p l i f i c a t i o n and rate enhancement, the feedback pathways o f the c l o t t i n g cascade have o n l y recently b e c o m e e l u c i d a t e d . A t the site o f an injury that leads to d i s r u p t i o n o f the integrity o f a b l o o d v e s s e l , a r a p i d and specific response must be e m p l o y e d to prevent e x c e s s i v e b l o o d loss and to restrict bacterial i n f e c t i o n [36]. In vivo, the f o r m a t i o n o f a f i b r i n c l o t requires a m i n i m u m o f five proteases: c o a g u l a t i o n factor X I ( F X I ) , c o a g u l a t i o n factor I X ( F I X ) , c o a g u l a t i o n factor V U ( F V I I ) , c o a g u l a t i o n factor X ( F X ) , and p r o t h r o m b i n . These e n z y m e s circulate i n the b l o o d stream at l o w concentrations i n i n a c t i v e , z y m o g e n forms. A c t i v a t i o n o f these z y m o g e n s requires the site-specific p r o t e o l y s i s o f one or m o r e peptide bonds, l i b e r a t i n g a free N terminus and p r o m o t i n g proteolytic a c t i v i t y (active forms o f the e n z y m e s are denoted w i t h a l o w e r case " a " , such as F X a ) [37]. L o c a l i z a t i o n o f the proteases to m e m b r a n e surfaces at the site o f i n j u r y is p r o v i d e d b y three co-factors: activated c o a g u l a t i o n factor V ( F V a ) , activated  14  c o a g u l a t i o n factor V I I I ( F V I I I a ) and tissue factor ( T F ) [38]. In c o m b i n a t i o n w i t h a p h o s p h o l i p i d b i l a y e r these co-factors promote the rate o f c l o t f o r m a t i o n ~ 1 0 - f o l d ( F i g u r e 6  1.7). D o w n - r e g u l a t i o n o f the p a t h w a y is m e d i a t e d i n part b y another protease, activated p r o t e i n C ( a P C ) , w h i c h c l e a v e s t w o o f the co-factors, F V a a n d F V I I I a , at specific p o s i t i o n s i n the p r o t e i n a n d inactivates t h e m [39]. T h r o m b i n plays a p i v o t a l r o l e i n the process as it activates p r o t e i n C , F V , a n d F V T I I as w e l l as a n u m b e r o f other s i g n a l i n g proteins that r e c r u i t cells a n d proteins to the site o f damage [40].  F i g u r e 1.7 O v e r v i e w o f vertebrate b l o o d c o a g u l a t i o n . F i v e proteases are i n v o l v e d i n the f o r m a t i o n o f a c r o s s - l i n k e d f i b r i n b l o o d c l o t (factors X I a , I X a , V i l a , X a , a n d t h r o m b i n ) . T h r e e accessory proteins (factors V i l l a a n d V a , a n d tissue factor ( T F ) ) are i n v o l v e d i n c o - l o c a l i z a t i o n o n a p h o s p h o l i p i d surface ( P L ) a n d c a t a l y t i c rate enhancement o f the entire process. P r o t e i n C ( P C ) is activated ( a P C ) b y t h r o m b i n and leads to i n h i b i t i o n o f the process b y c l e a v i n g the co-factors.  15  B i o c h e m i c a l characterization o f the p u r i f i e d c o m p o n e n t s o f the b l o o d c o a g u l a t i o n pathway  in vitro has  s h o w n that e a c h protease i n the p a t h w a y prefers to r e c o g n i z e and c l e a v e  a particular sequence o f a m i n o acids. Substrate s p e c i f i c i t y o f these proteases, h o w e v e r , is not e x t r e m e l y strict o w i n g to the requirement for the process to o c c u r r a p i d l y [41,42]. Substrate s p e c i f i c i t y is a c o m b i n a t i o n o f a d d i t i o n a l regions o f the e n z y m e that contribute to m o l e c u l a r r e c o g n i t i o n as w e l l as to the l o c a l architecture o f the substrate b i n d i n g site i n the active site o f the protease. Interactions between proteins are p r o v i d e d b y a d d i t i o n a l protein d o m a i n s i n the p o l y p e p t i d e sequence ( F i g u r e 1.8) [43]. C o n f i g u r a t i o n o f these d o m a i n s p r o v i d e s some clues to the e v o l u t i o n a r y history o f vertebrate b l o o d c o a g u l a t i o n .  Prothrombin FVII  ©000©^ ©©©©©^  Protein C  00000T£RoI]  Key: Apple Domain  ^ )  EGF Domain  Propeptide  Cf^j)  Fibronection Type I Domain  Gia Domain  ^jn^  Fibronectin Type II Domain  Kringle Domain  I PROT i Protease Domain  Signal Peptide  Figure 1.8  P r o t e i n d o m a i n s o f the vertebrate b l o o d c o a g u l a t i o n proteases.  16  T h r o u g h gene sequence analysis, gene d u p l i c a t i o n and d i v e r g e n c e o f the c o a g u l a t i o n proteases p r o b a b l y o c c u r r e d p r i o r to the emergence o f the vertebrate lineage. C o m p a r i s o n o f the p u b l i s h e d gene sequences f r o m a n u m b e r o f o r g a n i s m s r a n g i n g f r o m j a w l e s s vertebrates to h u m a n s s h o w s that the d o m a i n o r g a n i z a t i o n o f the c o a g u l a t i o n m a c h i n e r y is h i g h l y c o n s e r v e d i n a l l vertebrates [44]. S l i g h t variations are k n o w n to exist, h o w e v e r , i n c l u d i n g the absence o f the contact system ( F X I , F X I I , and k a l l i k r e i n ) i n fish. D o o l i t t l e has p r o p o s e d that the f o r m a t i o n o f the core o f the p a t h w a y o c c u r r e d r o u g h l y 4 5 0 m i l l i o n years ago [43]. In the i n t e r v e n i n g t i m e , a significant a m o u n t o f m o l e c u l a r e v o l u t i o n has taken p l a c e r e s u l t i n g i n n u m e r o u s changes to the gene sequence thereby r e s u l t i n g i n p a r a l l e l o p t i m i z a t i o n o f the relevant proteins for their p h y s i o l o g i c a l role.  1.8 Substrate Specificity of Serine Proteases H y d r o l y s i s o f a p o l y p e p t i d e c h a i n requires proper r e c o g n i t i o n , orientation and b i n d i n g o f the p o l y p e p t i d e b a c k b o n e . T h u s , the residues adjacent to the s c i s s i l e b o n d have a s i g n i f i c a n t i m p a c t o n the rate o f h y d r o l y s i s . N e a r l y 3 0 years ago, Schecter and B e r g e r d e s c r i b e d the substrate-protease interaction and their system has been adopted i n the literature ( F i g u r e 1.9)[45]. In this m o d e l the s c i s s i l e peptide b o n d is surrounded b y subsites o n the protease. Substrate a m i n o acids are termed P (for peptide) and the subsites o f the protease that interact w i t h t h e m are c a l l e d S (for subsite). Substrate residues e x t e n d i n g towards the N terminus o f the substrate are n u m b e r e d P 2 , P 3 , P 4 and so forth. C o n v e r s e l y , substrate residues e x t e n d i n g towards the C - t e r m i n u s are l a b e l e d P 2 ' , P 3 \ P 4 ' and o n w a r d s . R e g i o n s i n the protease are n u m b e r e d a c c o r d i n g to the substrate. F o r e x a m p l e , the P I residue is b o u n d i n the S I p o c k e t . T h e o r e t i c a l l y , a large a m o u n t o f v a r i a t i o n i n substrate s p e c i f i c i t y c a n result f r o m  17  the 2 0 p o s s i b i l i t i e s o f a m i n o a c i d side chains, and a w i d e d i v e r s i t y o f s p e c i f i c i t y is o b s e r v e d i n the nature.  Protease  Scissile Bond  Figure 1.9  Schecter & B e r g e r nomenclature o f protease s p e c i f i c i t y .  B r o a d l y specific proteases r e c o g n i z e and act at a site dictated b y a single a m i n o a c i d i n a p o l y p e p t i d e c h a i n whereas h i g h l y specific proteases r e c o g n i z e a short m o t i f c o n s i s t i n g o f three to eight a m i n o a c i d residues. T h e b i o l o g i c a l process i n w h i c h the protease is i n v o l v e d dictates the l e v e l o f s p e c i f i c i t y . D i g e s t i v e e n z y m e s f o u n d i n the gut, such as t r y p s i n and c h y m o t r y p s i n , r e c o g n i z e and c l e a v e p o l y p e p t i d e s based o n the presence o f a s i n g l e type o f a m i n o a c i d ( A r g / L y s a n d P h e / T r p / T y r , r e s p e c t i v e l y ) . H e n c e , a p o l y p e p t i d e substrate w o u l d t y p i c a l l y be degraded into m u l t i p l e fragments for further p r o c e s s i n g and absorption. I n contrast, a n u m b e r o f b i o l o g i c a l processes require m o r e specific p r o t e o l y s i s . A s m e n t i o n e d p r e v i o u s l y , the vertebrate b l o o d c l o t t i n g cascade relies o n the specific cleavage o f each m e m b e r o f the p a t h w a y to f u n c t i o n p r o p e r l y . A n u m b e r o f other b i o l o g i c a l processes require s i m i l a r l e v e l s o f s p e c i f i c i t y , p a r t i c u l a r l y w h e n used for s i g n a l i n g purposes such as h o r m o n e and c h e m o k i n e a c t i v a t i o n [46]. H o w e v e r , a trade o f f exists between the l e v e l o f substrate s p e c i f i c i t y and the catalytic e f f i c i e n c y o f the e n z y m e .  18  By demonstrating a h i g h degree o f s e l e c t i v i t y , the preferred substrate has a s l o w rate of association w i t h the e n z y m e . In turn, this generates a decreased catalytic rate r e l a t i v e to non-specific enzymes.  In vivo s u c h  a scenario is not often preferred and alternate m e c h a n i s m s  o f substrate s p e c i f i c i t y are e m p l o y e d . A s m e n t i o n e d i n the d i s c u s s i o n o f the b l o o d c o a g u l a t i o n system, a d d i t i o n a l p r o t e i n d o m a i n s are associated w i t h a protease d o m a i n to a i d p r o t e i n - . p r o t e i n interactions. T h u s , p h y s i o l o g y has p l a c e d a barrier o n the l e v e l o f s p e c i f i c i t y that a protease m i g h t possess. Proteases o f the b l o o d c o a g u l a t i o n system d i s p l a y a m a r k e d preference for certain a m i n o a c i d side chains i n the P I to P 4 p o s i t i o n s and h y d r o l y z e t h e m r a p i d l y . A l l c o a g u l a t i o n proteases have t r y p s i n - l i k e specific at the p r i m a r y ( P I ) p o s i t i o n and prefer to h y d r o l y z e peptide bonds o n the C - t e r m i n a l side o f A r g or L y s residues [47]. E x t e n d e d substrate s p e c i f i c i t y exists i n a l l c o a g u l a t i o n proteases i n the S 2 to S 4 b i n d i n g pockets. O n the basis o f their s p e c i f i c i t y , b o t h F X a and t h r o m b i n are w i d e l y used for the site-specific c l e a v a g e o f r e c o m b i n a n t proteins after I l e - G l u - G l y - A r g and L e u - V a l - P r o - A r g sequences i n a p o l y p e p t i d e c h a i n , r e s p e c t i v e l y [48]. S i m i l a r sequences to those preferred are k n o w n to be h y d r o l y z e d i n  vivo b y the  t w o proteases. K i n e t i c analysis o f these proteases has r e v e a l e d that both e n z y m e s  c a n effectively h y d r o l y z e other sequences o f a m i n o acids [41,49-51]. F o r e x a m p l e , F X a was i n i t i a l l y thought to have a strict preference for a s m a l l a m i n o a c i d side chains at P 2 ( G l y ) . H o w e v e r , large residues ( T r p , P h e ) at this p o s i t i o n are preferred  in vitro. T h r o m b i n c a n  be  g e n e t i c a l l y m a n i p u l a t e d to prefer one o f its t w o c l e a v a g e sites, w h i c h are different i n sequence and structure [52,53]. B a s e d o n these discrepancies, it seems p o s s i b l e to engineer at least this l e v e l o f substrate s p e c i f i c i t y into a b r o a d l y specific t r y p s i n - l i k e protease.  19  1.9 Genetic Manipulation of Serine Proteases G r e a t strides i n b i o t e c h n o l o g y have been made i n the past decade that a l l o w for the d e s i g n o f e n z y m e s w i t h desirable properties. E x a m p l e s o f successful m o d i f i c a t i o n s m a d e t h r o u g h p r o t e i n e n g i n e e r i n g i n c l u d e increased stability or a c t i v i t y at the extremes o f temperatures or p H , resistance to o x i d a t i o n , and stability i n non-aqueous e n v i r o n m e n t s [20]. M e t h o d s to introduce these properties i n v o l v e mutagenesis o f a target e n z y m e either t h r o u g h structure based d e s i g n or b y r a n d o m mutagenesis c o m b i n e d w i t h some f o r m o f genetic selection [54]. A r a t i o n a l d e s i g n strategy requires s i g n i f i c a n t amounts o f i n f o r m a t i o n , p a r t i c u l a r l y three d i m e n s i o n a l structures o f the i n i t i a l e n z y m e as w e l l as the k n o w l e d g e o f the r e g i o n s that w o u l d be i n v o l v e d . C o n v e r s e l y , a r a n d o m i z e d mutagenesis p r o c e d u r e c o m b i n e d w i t h selection or screening requires no i n f o r m a t i o n o f the sequence, structure or m e c h a n i s m and has been w i d e l y adopted for the alteration o f b i o c h e m i c a l properties o f e n z y m e s [55]. C e n t r a l to a l l forms o f p r o t e i n e n g i n e e r i n g is the c r e a t i o n o f a n o v e l p r o t e i n b y a d d i n g a n o v e l f u n c t i o n that w a s not possessed b y the target p r o t e i n . C r e a t i o n o f a h i g h l y specific protease suffers f r o m the p r a c t i c a l difficulties associated w i t h p r o d u c i n g a k i n e t i c a l l y w o r s e e n z y m e . A s m e n t i o n e d p r e v i o u s l y , for a protease to e x h i b i t a h i g h degree o f substrate s p e c i f i c i t y , some c o m p e n s a t i o n i n catalytic e f f i c i e n c y must be made. A n u m b e r o f studies have s h o w n that it is p o s s i b l e to s w i t c h substrate s p e c i f i c i t i e s amongst disparate m e m b e r s o f the protease f a m i l y . F o r e x a m p l e , H e d s t r o m demonstrated the c o n v e r s i o n o f a t r y p s i n - l i k e e n z y m e , w h i c h prefers P I A r g / L y s residues, into a c h y m o t r y p s i n l i k e e n z y m e that prefers P h e / T r p / T y r at P I [56-59]. T h e change i n specificity r e q u i r e d mutagenesis o f three surface l o o p s i n the e n z y m e as w e l l as a n u m b e r o f other p o i n t mutations. Importantly, the r e g i o n s c h a n g e d do not contact the substrate d i r e c t l y and the  20  r e s u l t i n g e n z y m e is i n e f f i c i e n t at c a t a l y z i n g the h y d r o l y s i s o f peptide bonds.  These  observations demonstrate the inherent c o m p l e x i t y o f d e s i g n i n g i m p r o v e d substrate s p e c i f i c i t y i n the c h y m o t r y p s i n f a m i l y . O t h e r m o d i f i c a t i o n s o f the substrate s p e c i f i c i t y i n the S I f a m i l y of proteases have also been successful ( T a b l e 1.3). A n u m b e r o f s p e c i f i c i t y determinants h a v e been u n c o v e r e d b y mutagenesis targeted to probe a d d i t i o n a l features o f the e n z y m e , s u c h as z y m o g e n p r o c e s s i n g and protease-inhibitor interactions [60-64]. T h e w e a l t h o f b i o c h e m i c a l and structural i n f o r m a t i o n a v a i l a b l e suggests the a b i l i t y to d e s i g n extended substrate s p e c i f i c i t y o f the S 2 to S 4 pockets.  Protease Trypsin  Engineered Property C o n v e r s i o n to elastase-like p r i m a r y  Ref. [65]  specificity Increased P I s p e c i f i c i t y towards A r g  [66]  side chains Increased P I s p e c i f i c i t y towards L y s  [67]  side chains S I ' E n g i n e e r i n g to favor basic residues  [68]  Kallikrein  Modification of S 2 binding pocket  [69]  FIXa  C o n v e r s i o n to F X a - l i k e extended  [70]  Thrombin  A l t e r a t i o n o f P 2 - P 4 preference creating  substrate s p e c i f i c i t y [52,53]  an anticoagulant protease  Table 1.3 Select e x a m p l e s o f successful p r o t e i n e n g i n e e r i n g o f serine proteases.  Streptomyces griseus  1.10  Trypsin  Streptomyces griseus t r y p s i n ( S G T ) was  initially purified from Pronase - a  c o m m e r c i a l preparation o f secreted proteases - and characterized o n the basis o f its h y d r o l y s i s o f N - b e n z o y l - L - a r g i n i n e e t h y l ester ( B A E E ) and c a s e i n and its i n h i b i t i o n b y s o y b e a n t r y p s i n a  i n h i b i t o r [71]. F u r t h e r characterization o f its s p e c i f i c i t y and i n h i b i t i o n i d e n t i f i e d S G T as a  21  t y p i c a l b r o a d s p e c i f i c i t y t r y p s i n - l i k e serine protease o f the S I f a m i l y that h y d r o l y z e s p o l y p e p t i d e c h a i n s o n the C - t e r m i n a l side o f basic residues ( A r g and L y s ) [72]. B a s e d o n sequence alignments, S G T is m o r e s i m i l a r to b o v i n e t r y p s i n than to other bacterial serine proteases [73,74]. T h e structure o f S G T w a s subsequently determined b y x - r a y c r y s t a l l o g r a p h y and refined to 1.7 A , and r e v e a l e d a three-dimensional f o l d that is also m o r e s i m i l a r to m a m m a l i a n serine proteases than bacterial proteases [75,76]. A l t h o u g h s i m i l a r i t i e s e x i s t at the sequence and structural l e v e l , S G T differs f r o m its m a m m a l i a n h o m o l o g u e s i n its r e d u c e d n u m b e r o f a m i n o a c i d insertions i n the p o l y p e p t i d e c h a i n w h e n a l i g n e d b y either sequence or structure ( A p p e n d i x A ) . M o r e o v e r , S G T contains o n l y three d i s u l f i d e bonds rather than the f i v e or s i x t y p i c a l l y o b s e r v e d i n the S I f a m i l y o f proteases. T h e s e differences suggest that S G T c o u l d be used as a m o d e l scaffold to study the substrate s p e c i f i c i t y a n d other properties demonstrated b y m a m m a l i a n proteases.  1.11 Statement of Hypothesis I f a l l substrate s p e c i f i c i t y determinants o f c o a g u l a t i o n factor X a are k n o w n , then their i n t r o d u c t i o n into  Streptomyces griseus t r y p s i n w i l l  result i n a protease w i t h s i m i l a r substrate  selectivity.  1.12 Objectives and Outline A l t h o u g h a w e a l t h o f data is a v a i l a b l e , several questions r e m a i n about the o v e r a l l m e c h a n i s m o f s p e c i f i c i t y o f serine proteases. A m o n g s t the p o o r l y characterized details o f serine proteases are the f l e x i b i l i t y o f the active site and its influence o n substrate s p e c i f i c i t y , the role o f water m o l e c u l e s i n the active site and the p o s s i b i l i t y o f d e s i g n i n g u l t r a - h i g h  22  s p e c i f i c i t y serine proteases that c a n be t a i l o r e d to desired reactions. In this study, S G T is d e v e l o p e d as a m o d e l for m a m m a l i a n serine protease s p e c i f i c i t y as it has s i m i l a r i t y i n b o t h sequence and structure, and is d e r i v e d f r o m a bacterial source that s h o u l d a l l o w for p r o d u c t i o n i n other bacteria and hence a l l o w genetic m o d i f i c a t i o n o f the protein. B a s e d o n the structural s i m i l a r i t i e s o b s e r v e d near and around the active site o f S .  griseus t r y p s i n c o m p a r e d  to m a m m a l i a n serine proteases, site-directed mutagenesis s h o u l d  p r o d u c e a c a t a l y t i c a l l y active protease w i t h the s p e c i f i c i t y o f factor X . F o u r questions are to be addressed i n this study: (1) D o e s the r e c o m b i n a n t S G T protein p r o d u c e d f r o m  Bacillus subtilis have  similar  e n z y m a t i c properties as the w i l d type protein? (2) W h a t mutations are r e q u i r e d to increase the p r i m a r y s p e c i f i c i t y o f the e n z y m e towards A r g side c h a i n s ? (3) W h a t p o i n t mutations or surface l o o p s near the active site confer a greater degree o f extended s p e c i f i c i t y i n the e n z y m e ? (4) W h a t are the c o m p l e t e requirements to c o n v e r t S G T to c o a g u l a t i o n factor X a - l i k e specificity? By e n g i n e e r i n g substrate s p e c i f i c i t y into a protease where little exists, a n u m b e r o f benefits w i l l result. A s m e n t i o n e d p r e v i o u s l y , proteases are used i n the site specific cleavage o f r e c o m b i n a n t proteins and the e n z y m e s used are c o s t l y due to their p r o d u c t i o n f r o m b l o o d . Proteases r e s u l t i n g f r o m a bacterial e x p r e s s i o n system w o u l d cost far less a n d facilitate increased usage. T h r o u g h the d e s i g n o f s p e c i f i c i t y s i m i l a r to a c o a g u l a t i o n factor, one c a n e x a m i n e the m e c h a n i s m s b y w h i c h the proteases generate s p e c i f i c i t y and the roles o f other regions o f the p o l y p e p t i d e that m i g h t be i n v o l v e d i n s p e c i f i c i t y . F o r e x a m p l e , n e a r l y a l l  23  c o a g u l a t i o n factor proteases have a specific s o d i u m b i n d i n g site and the b i n d i n g o f s o d i u m results i n a 3- to 5 - f o l d rate increase i n catalysis [77]. S o d i u m b i n d i n g i n t h r o m b i n also alters the substrate s p e c i f i c i t y o f the e n z y m e , yet d i s s e c t i o n o f this process has been l i m i t e d o w i n g to the interconnected relationship amongst b i o c h e m i c a l events w i t h i n the e n z y m e . D e v e l o p m e n t o f a r e c o m b i n a n t bacterial e x p r e s s i o n system for  S. griseus t r y p s i n  (Chapter 2) was a c r u c i a l obstacle to o v e r c o m e i n this research. U s i n g this e x p r e s s i o n system, the p r i m a r y s p e c i f i c i t y o f the protease was g e n e t i c a l l y engineered to favor A r g o v e r L y s side chains at the P I p o s i t i o n  (Chapter 3). Subsequent mutagenesis o f the S 2 to S 4  pockets o f S G T was c a r r i e d out to engineer c o a g u l a t i o n factor X a substrate s p e c i f i c i t y to the enzyme  (Chapter 4). O n the basis o f these results, a f r a m e w o r k for the p r o d u c t i o n o f n o v e l  proteases w i t h substrate specificities not o b s e r v e d i n nature is o u t l i n e d and other future directions are d i s c u s s e d  (Chapter 5).  24  Chapter 2. Recombinant Protein Expression of Streptomyces griseus Trypsin in Bacillus subtilis  2.1 Introduction D e v e l o p m e n t o f an efficient, cost effective and scaleable r e c o m b i n a n t p r o t e i n e x p r e s s i o n system is the first step i n p r o t e i n e n g i n e e r i n g . S o l u b l e , active and h i g h purity p r o t e i n must result f r o m an efficient e x p r e s s i o n system. A n u m b e r o f o r g a n i s m s have b e e n used for this purpose i n c l u d i n g those f r o m bacteria, f u n g i , yeast, and e u k a r y o t i c c e l l l i n e s . P r o t e i n e x p r e s s i o n i n l o w e r organisms, such as g r a m - p o s i t i v e and gram-negative bacteria, costs s i g n i f i c a n t l y less but the d r a w b a c k o f these systems is their i n a b i l i t y to p r o d u c e c o m p l e x proteins. L o n g e r p r o d u c t i o n times and h i g h e r costs are associated w i t h e u k a r y o t i c based systems; h o w e v e r , their use is u s u a l l y r e q u i r e d w h e n the protein to be p r o d u c e d is large (>60 k D a ) , has a c o m p l e x f o l d and contains d i s u l p h i d e bonds or requires post-translational m o d i f i c a t i o n (such as g l y c o s y l a t i o n ) . F o r protein e n g i n e e r i n g , bacterial, f u n g a l , or yeast e x p r e s s i o n hosts are t y p i c a l l y used due to their a m e n a b i l i t y to genetic m a n i p u l a t i o n and efficiency o f protein production. S u c c e s s f u l e x p r e s s i o n o f a r e c o m b i n a n t p r o t e i n i n l o w e r organisms requires a n u m b e r o f favorable b i o c h e m i c a l features. L o w t o x i c i t y , s i m p l e structural f o l d , l a c k o f d i s u l p h i d e b o n d s , l a c k o f post-translational m o d i f i c a t i o n , and s m a l l size tend to help p r o d u c t i o n . R e c o m b i n a n t e x p r e s s i o n is also s i g n i f i c a n t l y i n f l u e n c e d b y the properties o f the gene that encodes the p o l y p e p t i d e . O p t i m a l c o d o n usage and l a c k o f secondary structure have been s h o w n to be p r o b l e m a t i c i n the e x p r e s s i o n o f several proteins [78]. In m a n y instances,  25  h o w e v e r , h a v i n g favorable characteristics at the genetic and p r o t e i n l e v e l m a y still not result in successful e x p r e s s i o n and a great deal o f trial and error i n different systems is needed. B a c t e r i a l protein e x p r e s s i o n is w i d e l y p e r f o r m e d i n genetic m a n i p u l a t i o n ,  E. coli is u n r i v a l l e d i n the  Escherichia coli. A s a host  for  d i v e r s i t y and s i m p l i c i t y o f methods  established for genetic m a n i p u l a t i o n . P u r i f i c a t i o n and alteration o f D N A i n these g r a m negative bacteria is straightforward and h i g h l y r e p r o d u c i b l e . Indeed, m u c h o f the history o f m o l e c u l a r b i o l o g y is the result o f the study o f this o r g a n i s m . U n f o r t u n a t e l y ,  E. coli is not  as  adept at the p r o d u c t i o n o f f o r e i g n proteins. A n u m b e r o f attempts have been m a d e to engineer the g e n o m e o f this o r g a n i s m to increase its c a p a c i t y for r e c o m b i n a n t p r o t e i n p r o d u c t i o n , yet no u n i v e r s a l s o l u t i o n has been f o u n d [79]. F o r this reason a n u m b e r o f other bacteria have been investigated as alternatives i n c l u d i n g [83],  Pseuodomonads [84]  and  Bacilli [80,81], Lactobacilli [82], Streptomyctes  Caulobacter [85]. Importantly,  these alternatives have the  a b i l i t y to secrete heterologous proteins outside o f the c e l l . S e c r e t i o n eases subsequent p u r i f i c a t i o n , reduces the t o x i c i t y associated w i t h the p r o t e i n , a n d i m p r o v e s the rate o f f o r m a t i o n o f d i s u l p h i d e bonds. On the basis o f its bacterial source,  Streptomyces griseus t r y p s i n is suggested  as a  g o o d target for r e c o m b i n a n t protein e x p r e s s i o n and subsequent p r o t e i n e n g i n e e r i n g . S G T has m a n y features that are r e q u i r e d for successful protein e n g i n e e r i n g . T h e structure o f the e n z y m e has been determined at h i g h r e s o l u t i o n and a w e a l t h o f structure-function i n f o r m a t i o n has been d e s c r i b e d for h i g h l y s i m i l a r e n z y m e s [75,76,86]. A l t h o u g h it has a n u m b e r o f desirable properties for r e c o m b i n a n t e x p r e s s i o n such as its s m a l l size and s i m p l e f o l d , several features o f the gene and p r o t e i n c o u l d c o m p l i c a t e the p r o d u c t i o n o f the r e c o m b i n a n t protein. T r a n s l a t i o n o f the m R N A e n c o d i n g the p r o t e i n m a y be h a m p e r e d b y the h i g h g u a n i n e and  26  c y t o s i n e content o f the S G T gene ( 7 0 % ) . S e v e r a l authors have s h o w n that s u b - o p t i m a l c o d o n usage i n the first several c o d o n s c a n d r a m a t i c a l l y decrease protein e x p r e s s i o n [87,88]. In the r e d u c i n g e n v i r o n m e n t o f a bacterial c e l l , the nascent p o l y p e p t i d e c h a i n m a y have d i f f i c u l t y f o r m i n g the three d i s u l p h i d e bonds r e q u i r e d to stabilize the structure o f S G T [89]. L a s t l y , i f the protease is p r o d u c e d i n an active f o r m , it m a y degrade c o m p o n e n t s o f the c e l l as w e l l other S G T p o l y p e p t i d e s . T h e s e properties indicate that p r o d u c t i o n o f the r e c o m b i n a n t p r o t e i n m a y be difficult. O n c e p r o d u c e d b y an e x p r e s s i o n host, a r e c o m b i n a n t p r o t e i n must be p u r i f i e d to h o m o g e n e i t y . I n this process, some f o r m o f p r o t e i n capture to concentrate and c r u d e l y p u r i f y the p r o t e i n is t y p i c a l l y l i n k e d w i t h one or m o r e c h r o m a t o g r a p h i c separations. T h r o u g h o u t this procedure the loss o f protein, whether due to i n s t a b i l i t y or the a c t i v i t y o f contaminants, must be m i n i m i z e d . G i v e n the large b o d y o f literature o n serine proteases, p a r t i c u l a r l y t r y p s i n - l i k e proteases o f the c h y m o t r y p s i n f a m i l y , a n u m b e r o f reagents are a v a i l a b l e c o m m e r c i a l l y and c a n be used i n a variety o f p u r i f i c a t i o n methods. O n e particular advantage o f u s i n g a b a c t e r i a l e x p r e s s i o n host is the l a c k o f post-translational m o d i f i c a t i o n i n the target p r o t e i n w h i c h m i n i m i z e s sample heterogeneity i n the p r o t e i n p u r i f i e d . T h u s , r e c o m b i n a n t t r y p s i n - l i k e proteases d e r i v e d f r o m a bacterial source s h o u l d be e a s i l y p u r i f i e d i n h i g h y i e l d . T o b e g i n i n t r o d u c i n g substrate s p e c i f i c i t y into S G T , an efficient e x p r e s s i o n system was r e q u i r e d . I chose a bacterial e x p r e s s i o n system that w o u l d facilitate d o w n stream p r o c e s s i n g and future high-throughput studies.  B. subtilis is an  e x c e l l e n t e x p r e s s i o n system  for S G T due to its a b i l i t y to secrete active r e c o m b i n a n t p r o t e i n into the e x t r a c e l l u l a r e n v i r o n m e n t . C o m p a r i s o n o f the e n z y m a t i c properties and three d i m e n s i o n a l structure o f the p u r i f i e d protein to the n a t i v e l y d e r i v e d p r o t e i n f r o m  27  Streptomyces griseus s h o w  that b o t h  proteins are i d e n t i c a l . T h e s e studies p r o v i d e the basis for the subsequent e n g i n e e r i n g o f substrate s p e c i f i c i t y .  2.2 Materials & Methods 2.2.1 Plasmids, Bacterial Strains, and Growth Conditions  Escherichia coli was  g r o w n u s i n g standard methods [90].  B. subtilis strain  WB700  was g r o w n i n super-rich m e d i u m [91] or o n tryptose b l o o d agar base ( D i f c o ) at 3 7 ° C . F o r the  B. subtilis c a r r y i n g p l a s m i d p W B 9 8 0 [92], k a n a m y c i n  was added to a f i n a l concentration o f  10 p,g m l " i n b o t h l i q u i d and s o l i d m e d i a . 1  2.2.2 DNA manipulation Procedures for g e n o m i c (5.  griseus ( A T C C  10137)) and p l a s m i d D N A m a n i p u l a t i o n  were c a r r i e d out u s i n g established p r o t o c o l s [90]. P l a s m i d D N A was p u r i f i e d u s i n g a Q I A p r e p s p i n m i n i p r e p k i t ( Q i a g e n ) . E n z y m e s w e r e obtained f r o m N e w E n g l a n d B i o l a b s and R o c h e M o l e c u l a r B i o c h e m i c a l s . F o r P C R a m p l i f i c a t i o n o f the  SprT gene e n c o d i n g  S G T , the  f o l l o w i n g o l i g o n u c l e o t i d e s were d e s i g n e d to m a i n t a i n r e a d i n g frame o f the s a c B s i g n a l peptide present i n p l a s m i d p W B 9 8 0 : 5' - g g a a g c t t t t g c a G T C G T C G G C G G A A C C C G C G C G G - 3 ' 5' - g g t o t a g a t t a G A G C G T G C G G G C G G C C G A G G - 3 ' (restriction sites are u n d e r l i n e d , the  SprT gene specific  sequences are g i v e n i n upper case).  T h e P C R fragment was first c l o n e d into p B l u e s c r i p t K S + (Stratagene) and then s u b - c l o n e d into p W B 9 8 0 u s i n g the H i n d l l l and X b a l restriction e n z y m e sites c o n t a i n e d i n the o l i g o n u c l e o t i d e primers. T r a n s f o r m a t i o n o f  B. subtilis strain  28  W B 7 0 0 was p e r f o r m e d b y the  m e t h o d o f S p i z i z e n [93]. D N A sequence analysis o f the c l o n e d gene was p e r f o r m e d u s i n g the B i g D y e T e r m i n a t o r k i t and a n a l y z e d o n an A B I 3 7 0 0 D N A Sequencer ( A p p l i e d B i o s y s t e m s ) .  2.2.3 Protein Purification In order to p u r i f y the native and r e c o m b i n a n t protease to h o m o g e n e i t y , a p u r i f i c a t i o n strategy was d e v e l o p e d at l o w p H to m i n i m i z e autolysis. A f t e r centrifugation to r e m o v e c e l l u l a r debris (5,000 x g, 1 hr), r e c o m b i n a n t S G T was p u r i f i e d f r o m the supernatant o f 1 L o f  B. subtilis W B 7 0 0  culture. S e q u e n t i a l a m m o n i u m sulphate fractionation was c a r r i e d out at  3 0 % and 8 5 % saturation. T h e 8 5 % ( N H ^ S C M fraction pellet was resuspended i n 2 0 m M s o d i u m acetate buffer p H 4 . 5 , d i a l y z e d against the same buffer and a p p l i e d to a c o l u m n (15 c m x 1.5 c m ) o f S P Sepharose Fast F l o w ( A m e r s h a m P h a r m a c i a ) . A f t e r extensive w a s h i n g w i t h 2 0 m M s o d i u m acetate buffer c o n t a i n i n g 50 m M N a C I , p H 4.5, the b o u n d proteins were eluted w i t h 2 0 m M s o d i u m acetate buffer, p H 4 . 5 , c o n t a i n i n g 150 m M N a C I . T h e active fractions were p o o l e d and a p p l i e d to a B e n z a m i d i n e Sepharose 4 Fast F l o w c o l u m n (8 c m x 0.75 c m ) ( A m e r s h a m P h a r m a c i a ) . T h e c o l u m n was w a s h e d w i t h 2 0 m M s o d i u m acetate buffer c o n t a i n i n g 5 0 0 m M N a C I , p H 4.5, and the e n z y m e was eluted i n the same buffer c o n t a i n i n g i n a d d i t i o n 4 0 m M b e n z a m i d i n e H C 1 ( S i g m a ) . F r a c t i o n s c o n t a i n i n g active protease w e r e p o o l e d , concentrated and d i a l y z e d against 10 m M T r i s - H C I buffer c o n t a i n i n g 150 m M N a C I and 2 0 m M C a C l , p H 7.6 u s i n g a 10,000 N M W L Ultrafree-4 centrifugal filter unit ( M i l l i p o r e ) . G e l 2  filtration through a c o l u m n (45 c m x 0.75 c m ) o f Sephadex G - 7 5 ( A m e r s h a m P h a r m a c i a ) was p e r f o r m e d u s i n g 10 m M T r i s - H C I c o n t a i n i n g 150 m M N a C I and 2 0 m M C a C l , p H 7.6. 2  S i m i l a r l y , native S G T was isolated f r o m 1 g o f e x t r a c e l l u l a r filtrate f r o m  S. griseus ( S i g m a ) .  T h e f i n a l p r o t e i n concentration was determined b y U V absorbance at 2 8 0 n m , u s i n g the  29  e x t i n c t i o n coefficient 3 7 , 1 0 0 M "  1  c m " [94] or b y a B C A p r o t e i n assay k i t (Pierce). A c t i v e site 1  titration was p e r f o r m e d u s i n g 4 - n i t r o p h e n y l p ' - g u a n i d i n o b e n z o a t e and a standard c u r v e o f p n i t r o p h e n o l ( S i g m a ) . S o d i u m d o d e c y l s u l p h a t e p o l y a c r y l a m i d e g e l electrophoresis and C o o m a s s i e B l u e staining were p e r f o r m e d a c c o r d i n g to standard procedures [90]. N - t e r m i n a l p r o t e i n m i c r o s e q u e n c e analysis was p e r f o r m e d b y the U n i v e r s i t y o f V i c t o r i a - G e n o m e B C P r o t e o m i c s C e n t r e ( V i c t o r i a , C a n a d a ) . E l e c t r o s p r a y - m a s s spectrometry was c a r r i e d out o n a P E - S c i e x A P I 3 0 0 triple q u a d r u p o l e mass spectrometer ( S c i e x ) e q u i p p e d w i t h an Ionspray i o n source. T h e mass spectrometry was p e r f o r m e d b y D r . S. H e i n the W i t h e r s laboratory (Dept. of Chemistry, U B C ) .  2.2.4 Kinetic Analysis K i n e t i c analysis was p e r f o r m e d i n 10 m M T r i s - H C I buffer c o n t a i n i n g 150 m M N a C I , 2 0 m M C a C l , and 0.1 % P E G 8 0 0 0 , p H 7.6. A standard o f 7 - a m i n o 4 - m e t h y l c o u m a r i n 2  ( A M C ) was u s e d to quantify the rates o f h y d r o l y s i s o f the f l u o r o g e n i c substrates T o s - G l y - P r o A r g - A M C and T o s - G l y - P r o - L y s - A M C ( B a c h e m ) . A m i n i m u m o f six substrate concentrations r a n g i n g f r o m 1 to 5 0 \iM was used. T h e f i n a l concentration o f the e n z y m e i n e a c h assay was 0.5 n M . N o n - l i n e a r regression o f the i n i t i a l r e a c t i o n rates and c a l c u l a t i o n o f the k i n e t i c parameters were p e r f o r m e d u s i n g the G r a p h p a d P r i s m 3.0 software ( G r a p h p a d ) .  2.2.5 Crystallization In p r e v i o u s studies, crystals o f the native S G T were obtained through batch c r y s t a l l i z a t i o n u s i n g ( N H ) S 0 4 [75,76]. In the current study, proteins were c r y s t a l l i z e d u s i n g 4  2  s i m i l a r c o n d i t i o n s (10-15 m g / m L p r o t e i n , 1.5 M ( N H ^ S O ^ 10 m M c a l c i u m acetate, p H 6.2)  30  except h a n g i n g drop v a p o r d i f f u s i o n was u t i l i z e d w h e r e the r e s e r v o i r c o n t a i n e d 1.55 M  (NH ) S04. C r y s t a l s appeared i n t w o to three w e e k s to d i m e n s i o n s o f a p p r o x i m a t e l y 0.3 x 0.3 4  2  x 0.3 m m . D a t a w e r e c o l l e c t e d at 100 K ( O x f o r d C r y o s t r e a m ) w i t h a M a r 3 4 5 detector m o u n t e d o n a R i g a k u R U - 2 0 0 X - r a y generator (50 k V , 100 m A ) w i t h O s m i c f o c u s i n g m i r r o r s . C r y s t a l s were s o a k e d b r i e f l y i n 2 0 % g l y c e r o l , 2.2 M  (NH )2S04for c r y o p r o t e c t i o n 4  p r i o r to data c o l l e c t i o n . D a t a were processed u s i n g the H K L p a c k a g e and refined u s i n g C N S v e r s i o n 1.1 i n c o m b i n a t i o n w i t h X t a l v i e w [95-97]. T h e p r e v i o u s l y reported native S G T structure ( P D B entry 1 S G T ) was u s e d as a m o d e l for r i g i d b o d y refinement o f the structure [76].  2.3 Results and Discussion 2.3.1 Production and Purification of Recombinant SGT P r e v i o u s studies demonstrated the a b i l i t y to p r o d u c e s o l u b l e t r y p s i n - l i k e e n z y m e s i n the p e r i p l a s m i c space o f  E. coli [98-100]. I n o u r experiments,  however,  E. coli was  incapable  o f generating s o l u b l e S G T despite u s i n g a variety o f p l a s m i d constructs i n a n u m b e r o f bacterial host strains. T h e presence o f three d i s u l f i d e b o n d s , the h i g h G + C % content o f the  SprT gene ( 7 0 % )  and the t o x i c i t y o f the r e c o m b i n a n t p r o t e i n are p o s s i b l e reasons for the l a c k  of p r o d u c t i o n o f r e c o m b i n a n t proteins i n  subtilis W B 7 0 0 ,  E. coli [78,88]. T o o v e r c o m e these l i m i t a t i o n s B.  a strain that is deficient i n seven proteases, was u s e d to p r o d u c e sufficient  y i e l d s o f r e c o m b i n a n t S G T for k i n e t i c and structural analysis [101]. U n l i k e  E. coli, B. subtilis  is capable o f secreting proteins into the e x t r a c e l l u l a r e n v i r o n m e n t , w h i c h facilitates r a p i d detection, p u r i f i c a t i o n and analysis o f r e c o m b i n a n t proteins.  31  S e c r e t i o n o f proteins into the e x t r a c e l l u l a r m e d i u m is facilitated b y the presence o f a single p l a s m a m e m b r a n e i n the g r a m - p o s i t i v e b a c t e r i u m  B. subtilis. F o l l o w i n g  translation b y  the r i b o s o m e , a nascent p o l y p e p t i d e c h a i n is targeted for secretion b y the presence o f a c l e a v a b l e a m i n o - t e r m i n a l s i g n a l peptide. O n the basis o f g e n o m e sequence analysis, o v e r 3 0 0 proteins ( - 7 . 3 % o f all genes) have been postulated for secretion i n  B. subtilis [ 1 0 2 - 1 0 4 ] .  The  a b i l i t y to secrete s u c h a w i d e d i v e r s i t y o f proteins b y this o r g a n i s m has been used for the p r o d u c t i o n o f a n u m b e r o f r e c o m b i n a n t proteins [105-107]. P r o t e i n secretion i n a l l bacteria, i n c l u d i n g  B. subtilis, is p r i m a r i l y due  to the  ATP-  dependent S e c p a t h w a y . R e c o g n i t i o n o f the s i g n a l peptide is m e d i a t e d b y the s i g n a l r e c o g n i t i o n particle p r o t e i n c o m p l e x , w h i c h shuttles the u n f o l d e d p r o t e i n to the c e l l m e m b r a n e [108]. R e m o v a l o f the s i g n a l peptide occurs as the denatured p r o t e i n translocates across the c e l l m e m b r a n e and is t y p i c a l l y c a r r i e d out b y the type I s i g n a l peptidase S i p S i n B .  subtilis [109].  In o u r e x p r e s s i o n system, secretion o f S G T into the e x t r a c e l l u l a r e n v i r o n m e n t  was m e d i a t e d b y fusing the gene to the s i g n a l peptide sequence o f levansucrase ( S a c B ) ( F i g u r e 2.1). T h u s , the N - t e r m i n u s o f the p r o t e i n is not accessible to the active site and the protease is i n a c t i v e u n t i l it is secreted f r o m the c e l l . C l e a v a g e after the A l a - P h e - A l a sequence at the j u n c t i o n o f the f u s i o n p r o t e i n b y S i p S generates the correct N - t e r m i n u s . S G T c a n then f o l d i n the c o m p a r a t i v e l y n o n - r e d u c i n g e n v i r o n m e n t outside o f the c e l l . B y m i m i c k i n g the native o r g a n i s m for the p r o d u c t i o n o f the r e c o m b i n a n t S G T , w e have d e v e l o p e d an efficient and n o v e l e x p r e s s i o n system for the p r o d u c t i o n o f t r y p s i n - l i k e e n z y m e s .  32  Xbal (4376)  Replicase  Kanamycin nucleotidyltransferase  F i g u r e 2.1 P l a s m i d m a p o f S G T gene c l o n e d into p W B 9 8 0 for protein e x p r e s s i o n i n  B.  subtilis.  recombinant  T h e gene was c l o n e d to m a i n t a i n the r e a d i n g  frame o f the S a c B s i g n a l peptide w h i c h facilitates secretion o f the r e c o m b i n a n t protein into the e x t r a c e l l u l a r environment.  R e c o m b i n a n t protein y i e l d s o f >15 m g / L o f culture m e d i u m w e r e o b t a i n e d w i t h i n 2 4 hours o f g r o w t h at 37°C. T h e four step p u r i f i c a t i o n t y p i c a l l y p r o d u c e d 10-15 m g / L o f B.  subtilis culture  w i t h an o v e r a l l y i e l d o f 8 0 % (Table 2.1). F o u r separation techniques were  a p p l i e d to y i e l d the highest p u r i t y e n z y m e possible. T h e methods were c h o s e n based o n their c o m p a t i b i l i t y and m i l d c o n d i t i o n s . F o r e x a m p l e , an affinity c h r o m a t o g r a p h y step u s i n g soybean t r y p s i n i n h i b i t o r w a s f o u n d to b i n d S G T effectively. H o w e v e r , r e m o v a l o f the protein  33  f r o m this type o f c o l u m n r e q u i r e d p H 2.0 and it was feared that s u c h c o n d i t i o n s m a y destroy unstable mutants o f S G T .  Vol.  Total  Total  Specific  Purification  Yield  (mL)  Protein  Activity  (fold)  (%)  (mg)  (mmol/s)  Activity (pmol/s/mg)  1000  16000  59  4  1  100  (NH ) S0 precipitate  50  400  53  134  34  90  S P Sepharose  15  18.3  51  2774  694  87  Benzamidine  5  12.4  49  3856  964  81  4  12.2  48  3955  989  81  Media 4  2  4  Sepharose G-75 Superdex  Table 2.1 P u r i f i c a t i o n table for r e c o m b i n a n t S G T ( b S G T ) f r o m extracellular  supernatant.  Activity  was  measured  by  the  B .  subtilis  hydrolysis o f  the  c h r o m o g e n i c substrate B z - U e - G l u - A r g - p N A at 4 0 u M i n 10 m M T r i s - H C I , 2 0 m M C a C l , p H 7.6. 2  N a t i v e a n d r e c o m b i n a n t S G T were p u r i f i e d to h o m o g e n e i t y p r i o r to analysis. P u r i t y was assessed b y several different criteria. B y electrospray i o n i z a t i o n mass spectrometry, expected  and  observed  masses  for  the  native  and  recombinant  protein  were  the  within  e x p e r i m e n t a l error (23106.9 and 2 3 1 0 7 . 0 a m u , r e s p e c t i v e l y ) . F o r b o t h proteins the integrated data f r o m the m/z  +  fragments y i e l d e d a single u n a m b i g u o u s peak w i t h m i n i m a l b a c k g r o u n d  ( F i g u r e 2.2). In a d d i t i o n , w h e n the protease was treated w i t h p h e n y l m e t h a n e s u l f o n y l f l u o r i d e , S D S - P A G E s h o w e d a single b a n d o f the expected m o l e c u l a r w e i g h t ( F i g u r e 2.3). O n l y t w o autolytic fragments o f S G T were o b s e r v e d w h e n the protease was b o i l e d p r i o r to S D S - P A G E i n the absence o f a strong i n h i b i t o r .  34  A m i n o - t e r m i n a l sequence analysis r e v e a l e d that the w i l d - t y p e e n z y m e h a d a single u n a m b i g u o u s sequence N H - V a l - V a l - G l y - G l y - T h r - A r g c o r r e s p o n d i n g to the p u b l i s h e d S G T 2  sequence (12). T o g e t h e r w i t h the p r o t e i n assays and active site titration data, these results suggest that the f i n a l protein preparation was greater than 9 9 % pure.  BioSpec Reconstruct for +Q1: 5.92 min (6 scans) from BSGT 23107.0  1.80e5 cps  1.4e5H  21047.0 21637.0  24355.0  LA L< A ^ ^ J w a k l a ^ i i .. 21000  23000 Mass, amu  25000  Figure 2.2. E S - M S spectrum o f p u r i f i e d r e c o m b i n a n t S G T . T h e s i n g l e m/z peak at 2 3 , 1 0 7 . 0 a m u w i t h m i n i m a l b a c k g r o u n d indicates the h i g h p u r i t y o f the r e c o m b i n a n t protein.  35  kDa 97.4 66.2  A  45.0 31.0 21.5 14.4  F i g u r e 2.3 S D S - P A G E o f p u r i f i e d r e c o m b i n a n t S G T . L a n e A : b S G T (0.5 //g) inactivated w i t h P M S F prior to a d d i t i o n o f S D S - P A G E l o a d i n g buffer and b o i l i n g . L a n e B : b S G T (1.0 jug) w i t h o u t P M S F i n h i b i t i o n . T h e central lane o f the g e l contains l o w - r a n g e protein m o l e c u l a r mass standards ( B i o - R a d ) w h o s e masses are g i v e n o n the left-hand side o f the g e l . T h e g e l was stained w i t h Coomassie Brilliant Blue.  2.3.2 K i n e t i c A n a l y s i s T w o f l u o r o g e n i c peptide substrates, T o s - G l y - P r o - A r g - A M C and T o s - G l y - P r o - L y s A M C , were used to m o n i t o r the P I A r g : L y s preference o f the native and r e c o m b i n a n t proteases ( T a b l e 2.2). T h e k i n e t i c parameters o f the native a n d r e c o m b i n a n t w i l d - t y p e S G T are s i m i l a r to p r e v i o u s l y reported values u s i n g the same pair o f substrates [66]. O n the basis o f the s i m i l a r rates o f h y d r o l y s i s o f these peptide substrates, w e c a n c o n c l u d e that the r e c o m b i n a n t protein behaves i d e n t i c a l l y to the native protease.  36  SGT  bSGT  Tos-Gly-Pro-Arg-AMC  Tos-Gly-Pro-Lys-AMC  kcat  kcat / K m  kcat  K  SR/SK*  kcat / K m  m  (min")  (uM)  (min" u M " )  (min)  (u.M)  (min" u.M")  4880  2.3  2122.  1670  3.6  464  4.6  ±410  ±0.2  ± 120  ±0.4  4570  2.0  1520  3.2  475  4.8  ± 1210  ±0.2  ±60  ±0.2  2285  * S R / S K = ( T o s - G l y - P r o - A r g - A M C k ^ / K ) / ( T o s - G l y - P r o - L y s - A M C kc, / m  Table 2.2  P I a r g i n i n e to l y s i n e preference o f S G T e n z y m e s . A r g : L y s  K ) m  preference  w a s m e a s u r e d b y a m i d o l y t i c a c t i v i t y o f the native ( S G T ) , r e c o m b i n a n t S G T ( b S G T ) u s i n g t w o f l u o r o g e n i c peptides, T o s - G l y - P r o - A r g - A M C  and  Tos-Gly-  P r o - L y s - A M C . V a l u e s o b t a i n e d i n triplicate ± S . D .  2.3.3 Crystallization and Structure Determination X - r a y d i f f r a c t i o n data w e r e o b t a i n e d for the r e c o m b i n a n t w i l d - t y p e S G T at 1.5 A r e s o l u t i o n . D a t a c o l l e c t i o n a n d refinement statistics are g i v e n i n T a b l e 2 . 3 . T h e r e c o m b i n a n t protease c r y s t a l l i z e d i n the C 2 2 2 ] space g r o u p and c o n t a i n e d one m o l e c u l e p e r a s y m m e t r i c u n i t a n d a M a t t h e w s c o e f f i c i e n t o f 2.2 A / D a ( T a b l e 2.3). T h e structure w a s deposited i n the 3  P D B database as l O S S . D u r i n g refinement, l o w R s t a n d R f cry  r e e  v a l u e s w e r e o b t a i n e d (0.19 and  0.22). T h e s e w e r e a c c o m p a n i e d b y e x c e l l e n t stereochemistry i n d i c a t i n g a h i g h q u a l i t y m o d e l . I n s p e c t i o n o f the R a m a c h a n d r a n p l o t r e v e a l e d that a l l n o n - g l y c i n e b a c k b o n e atoms are i n a l l o w e d r e g i o n s , w i t h o n l y A s n l 7 8 a d o p t i n g a c o n f o r m a t i o n i n the g e n e r o u s l y a l l o w e d r e g i o n ( F i g u r e 2.4). T h e o v e r a l l B - f a c t o r s for the p o l y p e p t i d e atoms w e r e l o w ( - 1 3 A ) , and r e g i o n s 2  w i t h h i g h B - f a c t o r s w e r e l i m i t e d to s o l v e n t e x p o s e d r e g i o n s that are not i n v o l v e d i n c r y s t a l p a c k i n g . T h e structure o f S G T is the highest q u a l i t y reported to date l i k e l y due to the  37  decreased radiation d a m a g e as the present crystals were a n a l y z e d under c r y o g e n i c c o n d i t i o n s w i t h a shorter c o l l e c t i o n time.  Phi  (degrees)  F i g u r e 2.4 R a m a c h a n d r a n p l o t o f the crystal structure o f r e c o m b i n a n t w i l d - t y p e S G T . A l l n o n - g l y c i n e residues are i n the a l l o w e d c o n f o r m a t i o n . T h e p l o t w a s c a l c u l a t e d using  PROCHECK[110]  38  Data collection R e s o l u t i o n (A)  1.5 ( 1 . 5 5 - 1.65)  Total Observations  25923  C o m p l e t e n e s s (%)  84.1 (74.1)  Average redundancy  2.8  I/oT  2 0 . 4 (5.1)  Rmerge (%)  4.1 (18.2)  R e f i n e m e n t statistics Space g r o u p  C222i  C e l l d i m e n s i o n s (a,b,c) *  5 0 . 0 4 , 6 9 . 8 2 , 119.65  M o l e c u l e s per a s y m m e t r i c u n i t  1  Rcryst  0.196  Rfree  0.222  P r o t e i n atoms*  1623  S o l v e n t atoms per a s y m m e t r i c u n i t  211  A v e r a g e B - f a c t o r for protein (A )  12.9  A v e r a g e B - f a c t o r for water (A )  21.0  Occupancy of C a  0.53 (14.98)  2  2  2 +  (B A ) 2  B o n d length deviations (A)  0.007  B o n d angle deviations (°)  1.4  a = P = y = 9 0 ° ; i n c l u d i n g alternate side c h a i n c o n f o r m a t i o n s  Table 2.3 D a t a c o l l e c t i o n and refinement statistics o f w i l d - t y p e r e c o m b i n a n t S G T . Statistics for the highest r e s o l u t i o n s h e l l are g i v e n i n parentheses.  39  2.3.4 Comparison of the recombinant and Pronase-derived crystal structure A l l a m i n o a c i d residues i n the m o d e l o f the r e c o m b i n a n t S G T were c l e a r l y i d e n t i f i e d and p o s i t i o n e d . I n 1 S G T [76], several residues h a d w e a k or absent s i d e - c h a i n density. In the present structure, m o s t o f these densities were c l e a r l y r e s o l v e d , a l t h o u g h several residues l a c k e d density i n the t e r m i n a l atoms o f their side-chains ( T h r 2 0 , G l n 7 5 , L y s 8 2 , T h r 9 8 , S e r 2 3 6 , and A r g 2 4 3 ) i n d i c a t i n g disorder o f these solvent e x p o s e d atoms. R e s i d u e s 77 and 7 9 were m o d e l e d as G l y and A l a i n 1 S G T but are t w o Ser residues b y D N A sequence analysis [74]. T h e s e Ser residues were c l e a r l y r e s o l v e d i n the current electron density m a p . T h e electron density o f one sulphate i o n was o b s e r v e d i n the o x y a n i o n h o l e o f the substrate b i n d i n g site and was i n c l u d e d i n the structure. T h e p o s i t i o n o f this sulphate is c o n s e r v e d i n a n i o n i c s a l m o n t r y p s i n ( P D B entry 1 B I T ) , b o v i n e t r y p s i n ( 1 T L D ) , and p o r c i n e pancreatic elastase ( 3 E S T ) [111-113]. A l t e r n a t e c o n f o r m a t i o n s were o b s e r v e d for G l n l 9 2 , w h i c h either points into the solvent or forms a p a i r o f h y d r o g e n bonds w i t h the b a c k b o n e N H o f G l y l 4 8 o f an adjacent S G T m o l e c u l e . I n 1 S G T the same residue was noted as h a v i n g h i g h m o b i l i t y [76]. D i f f e r e n c e s i n the C a  + 2  b i n d i n g site i n the r e c o m b i n a n t crystal structure are d i s c u s s e d i n  C h a p t e r 3. T h e o v e r a l l differences b e t w e e n the native ( 1 S G T ) and w i l d - t y p e r e c o m b i n a n t e n z y m e are m i n o r ( F i g u r e 2.5), w i t h a root m e a n square d e v i a t i o n o f a l l 8 9 2 atoms i n the C  a  b a c k b o n e o f 0.27 A . T h e largest d e v i a t i o n i n b a c k b o n e occurs at the 174-loop w h e r e peptide b o n d o f A l a l 7 7 a adopts a 1 8 0 ° rotation c o m p a r e d to the native structure.  40  F i g u r e 2.5 S u p e r i m p o s i t i o n o f the C traces o f native a n d r e c o m b i n a n t w i l d - t y p e S G T . a  T h e t w o structures ( P D B I D 1 S G T and 1 0 S 8 ) are i n d i s t i n g u i s h a b l e as i n d i c a t e d b y the s m a l l r.m.s d e v i a t i o n between the C b a c k b o n e atoms o f the t w o structures (0.27 A ) . u  2.3.5 W i l d - t y p e N a t i v e a n d R e c o m b i n a n t S G T S u b s t r a t e B i n d i n g A negatively c h a r g e d residue ( D 1 8 9 ) is present i n the S I p o c k e t and confers the p r i m a r y specificity o f S G T a n d other t r y p s i n - l i k e e n z y m e s towards p o s i t i v e l y c h a r g e d A r g or L y s side chains [114]. B a s e d o n the structure o f 1 S G T , D 1 8 9 is l o c a t e d at the base o f a narrow c y l i n d r i c a l cleft that c a n a c c o m m o d a t e these side c h a i n s [76]. T h e s l i g h t preference for A r g o v e r L y s i n this p o c k e t is due to the requirement for a b r i d g i n g water m o l e c u l e between the shorter l y s y l - s i d e c h a i n a n d D 1 8 9 [66]. T h e y - O H o f residue T 1 9 0 interacts directly w i t h the substrate v i a h y d r o g e n b o n d i n g . B o t h L y s a n d A r g side c h a i n s adopt  41  favorable c o n f o r m a t i o n s for interaction w i t h D 1 8 9 and the h y d r o x y l g r o u p o f T 1 9 0 . K i n e t i c analysis o f the native and r e c o m b i n a n t S G T proteases demonstrated an A r g : L y s preference o f 4:1. E x t e n d e d substrate s p e c i f i c i t y is l a r g e l y absent i n a l l b r o a d l y specific t r y p s i n - l i k e e n z y m e s . A c c e s s i b i l i t y o f the catalytic triad i n S G T is not h i n d e r e d b y the structure o f the active site. C r y s t a l structures o f other t r y p s i n - l i k e e n z y m e s have demonstrated that the peptide b a c k b o n e o f substrate residues P I to P 3 forms an anti-parallel (3-sheet w i t h residues 214 to 2 1 6 i n the e n z y m e [24]. A n a l y s i s o f the substrate s p e c i f i c i t y o f the S 2 to S 4 pockets i n b o v i n e t r y p s i n has s h o w n an absence for preference o f any side c h a i n at these positions [115,116]. T h e structure o f S G T suggests an i d e n t i c a l m o d e o f substrate b i n d i n g and an absence o f substrate s p e c i f i c i t y i n the S 2 to S 4 pockets.  2.4 Conclusions A n o v e l e x p r e s s i o n system for the p r o d u c t i o n o f r e c o m b i n a n t S G T has been d e v e l o p e d using  B. subtilis. H i g h  purity protease resulted f r o m a four-step p u r i f i c a t i o n p r o t o c o l . T h e  r e c o m b i n a n t p r o t e i n demonstrates i d e n t i c a l b i o c h e m i c a l a n d structural properties to the  Streptomyces d e r i v e d protease. O n the  basis o f the h i g h l e v e l o f p r o d u c t i o n , purity, and a b i l i t y  to c r y s t a l l i z e the p r o t e i n , the r e c o m b i n a n t p r o t e i n is h i g h l y a m e n a b l e for e n g i n e e r i n g extended substrate s p e c i f i c i t y into the e n z y m e .  42  Chapter 3. Engineering the Primary Substrate Specificity of Streptomyces griseus Trypsin  3.1 Introduction Substrate s p e c i f i c i t y is a k e y c o n c e p t i n the analysis o f serine proteases. Sequence analysis studies s h o w that the S I f a m i l y o f t r y p s i n - l i k e e n z y m e s l i k e l y e v o l v e d f r o m a c o m m o n ancestral gene [117]. T h e c u l m i n a t i o n o f i n c r e m e n t a l e v o l u t i o n a r y steps l e d to the appearance o f a n u m b e r o f h i g h l y s p e c i f i c proteases such as those f o u n d i n the vertebrate b l o o d c o a g u l a t i o n cascades. T h e s e proteases f u l f i l l regulatory roles i n c e l l u l a r processes that are distinct f r o m their m o r e p r i m i t i v e roles as degradative and protective e n z y m e s [118]. M u c h data have been c o l l e c t e d o n the specificity determinants o f serine proteases, r e s u l t i n g i n a c l a s s i f i c a t i o n system based o n their p r i m a r y s p e c i f i c i t y ( S I p o c k e t ) . T h e s p e c i f i c i t y o f t r y p s i n - l i k e e n z y m e s at the S I p o c k e t is l a r g e l y defined b y the presence o f a n e g a t i v e l y c h a r g e d side c h a i n at p o s i t i o n 189 [114]. O p t i m a l b i n d i n g o f the p o s i t i v e l y c h a r g e d substrate ( A r g or L y s side chains) to residue 189 is m e d i a t e d b y residue 190 [66,119]. I n t r y p s i n - l i k e serine proteases, p o s i t i o n 190 is o c c u p i e d b y a l i m i t e d n u m b e r o f a m i n o acids. D e g r a d a t i v e proteases w i t h l o w p r i m a r y s p e c i f i c i t y ( A r g : L y s preference o f 4:1) d i s p l a y G i n , T h r or Ser at p o s i t i o n 190, whereas proteases w i t h h i g h p r i m a r y s p e c i f i c i t y ( A r g : L y s preferences greater than 7:1) c o n t a i n A l a or Ser at this p o s i t i o n [120]. P r e v i o u s studies have s h o w n that mutagenesis o f p o s i t i o n 190 c a n be used to m a n i p u l a t e the substrate s p e c i f i c i t y o f t r y p s i n to favor c l e a v a g e after either A r g or L y s side chains [66,67,119]. S i m i l a r to degradative vertebrate t r y p s i n - l i k e e n z y m e s , S G T demonstrates a p r i m a r y substrate preference o f A r g L y s o f 4 : 1 . In the p r e v i o u s chapter I d e s c r i b e d the p r o d u c t i o n o f  43  f u l l y active r e c o m b i n a n t S G T f r o m  B. subtilis. U s i n g  this system, mutants o f S G T were  constructed w i t h altered preference for arginine to l y s i n e ( A r g : L y s ) . M u t a t i o n s were d e s i g n e d to m i m i c those f o u n d i n other t r y p s i n - l i k e e n z y m e s . S G T mutant T 1 9 0 P is c o n s i d e r a b l y m o r e active and less A r g - s p e c i f i c w h e n c o m p a r e d w i t h the p r e v i o u s l y p u b l i s h e d S 1 9 0 P m u t a t i o n created i n rat a n i o n i c t r y p s i n [67]. K i n e t i c and structural analysis o f the mutant protease s h o w s that both the a c t i v i t y and s p e c i f i c i t y o f the e n z y m e is affected b y residues s u r r o u n d i n g residue 190. T h e s e results further o u r understanding o f the p r i m a r y substrate s p e c i f i c i t y o f t r y p s i n - l i k e e n z y m e s . B a s e d o n the ease of p r o d u c t i o n and p u r i f i c a t i o n o f the r e c o m b i n a n t p r o t e i n i n o u r  B. subtilis e x p r e s s i o n  system,  SGT is an i d e a l scaffold for the i n t r o d u c t i o n o f a d d i t i o n a l mutations to enhance the substrate s p e c i f i c i t y o f the S 2 to S 4 b i n d i n g pockets.  3.2 Materials & Methods 3.2.1 DNA Manipulation and Protein Purification U s i n g the p r e v i o u s l y d e s c r i b e d S G T gene c l o n e d into p B l u e s c r i p t K S + p l a s m i d , mutagenesis was p e r f o r m e d o n the gene u s i n g a Q u i k C h a n g e site-directed mutagenesis k i t (Stratagene) as d e s c r i b e d b y the manufacturer. O l i g o n u c l e o t i d e s used for mutagenesis are p r o v i d e d i n T a b l e 3.1. D N A sequence analysis o f the c l o n e d gene and mutants was p e r f o r m e d u s i n g the B i g D y e T e r m i n a t o r k i t and a n a l y z e d o n an A B I 3 7 0 0 D N A Sequencer ( A p p l i e d B i o s y s t e m s ) . M u t a n t S G T genes were s u b - c l o n e d into p l a s m i d p W B 9 8 0 and transformed into  B. subtilis W B 7 0 0  as described p r e v i o u s l y . M u t a n t S G T proteins were expressed and p u r i f i e d  in an i d e n t i c a l m a n n e r as the w i l d - t y p e .  44  Mutation  Oligon  ucleotide  T190A  5' - G G C G T C G A C G C C T G C C A G G G T - 3 '  T190P  5' - G G C G T C G A C C C C T G C C A G G G T - 3 '  T190V  5' - G G C G T C G A C T T C T G C C A G G G T - 3 '  T190V  5' - G G C G T C G A C G T C T G C C A G G G T - 3 '  Table 3.1 O l i g o n u c l e o t i d e s used to mutate residue 190 i n the S G T gene. T h e reverse c o m p l e m e n t sequences o f these o l i g o n u c l e o t i d e s were also used i n the mutagenesis.  3.2.2 Kinetic Analysis K i n e t i c analysis was p e r f o r m e d i n 10 m M T r i s H C 1 buffer c o n t a i n i n g 150 m M N a C I , 2 0 m M C a C l , and 0.1 % P E G 8 0 0 0 , p H 7.6. A standard o f 7 - a m i n o 4 - m e t h y l c o u m a r i n 2  ( A M C ) was used to quantify the rates o f h y d r o l y s i s o f the f l u o r o g e n i c substrates T o s - G l y - P r o A r g - A M C and T o s - G l y - P r o - L y s - A M C ( B a c h e m ) . A m i n i m u m o f s i x substrate concentrations r a n g i n g f r o m 1 to 5 0 0 u . M w a s used. E n z y m e concentrations ranged f r o m 0.5 to 10 n M . B e n z a m i d i n e concentrations ranged f r o m 5 to 2 0 0 n M . N o n - l i n e a r regression o f the i n i t i a l reaction rates and c a l c u l a t i o n o f the k i n e t i c parameters w e r e p e r f o r m e d u s i n g the G r a p h p a d P r i s m 3.0 software ( G r a p h p a d ) .  3.2.3 Crystallization and Structure Determination C r y s t a l l i z a t i o n c o n d i t i o n s for the T 1 9 0 P mutant o f S G T w e r e s i m i l a r to the w i l d - t y p e p r o t e i n ; h o w e v e r 25 m M b e n z a m i d i n e was i n c l u d e d i n the sample buffer. C r y s t a l s appeared i n t w o to three w e e k s to d i m e n s i o n s o f 0.3 x 0.3 x 0.3 m m . D a t a c o l l e c t i o n and structure refinement were i d e n t i c a l to those used for the w i l d - t y p e r e c o m b i n a n t structure. E l e c t r o n  45  density o f the i n h i b i t o r i n the S I p o c k e t o f the mutant e n z y m e was evident, and was i n c l u d e d i n the final m o d e l .  3.3 Results and Discussion 3.3.1 Production of SGT Mutants E x p r e s s i o n l e v e l s o f the four mutant proteases were c o m p a r a b l e to the w i l d - t y p e S G T p r o t e i n suggesting that the mutations were not detrimental to the o v e r a l l stability and f o l d i n g o f the proteases. T o ensure accurate k i n e t i c s , the c o m p l e t e m e t h o d u s i n g the four-step p u r i f i c a t i o n p r o t o c o l was a p p l i e d to each p r o t e i n . E S - M S analysis o f the r e c o m b i n a n t proteases demonstrated the presence o f the m u t a t i o n , as w e l l as h i g h p u r i t y o f the s a m p l e ( T a b l e 3.2).  Theoretical Mass (amu)  Observed Mass (amu)  Difference (amu)  T190A  23077.0  23079.0  2~0  T190P  23103.0  23098.8  4.2  T190S  23093.0  23090.0  3.0  T190V  23107.1  23102.0  5.1  Table 3.2 E S - M S analysis o f the four mutants o f S G T .  3.3.2 Kinetic Analysis T w o f l u o r o g e n i c peptide substrates, T o s - G l y - P r o - A r g - A M C and T o s - G l y - P r o - L y s A M C , were used to m o n i t o r the P I A r g : L y s preference o f r e c o m b i n a n t and mutant proteases ( T a b l e 3.3). V a l u e s for the native and w i l d - t y p e r e c o m b i n a n t S G T are p r o v i d e d i n the table  46  for reference.  M u t a n t s T 1 9 0 P a n d T 1 9 0 A demonstrated a s i g n i f i c a n t increase i n P I A r g : L y s  preference o v e r the w i l d - t y p e e n z y m e o f 18:1 a n d 8:1, r e s p e c t i v e l y . A l l four mutants s h o w e d increased K  m  values f o r the substrates tested suggesting that the S I p o c k e t o f S G T i s  o p t i m i z e d f o r substrate b i n d i n g , a feature that has been o b s e r v e d i n other t r y p s i n - l i k e e n z y m e s [66,67,119]. A s i m i l a r trend o f f o l d differences for the K i v a l u e o f the s m a l l m o l e c u l e i n h i b i t o r b e n z a m i d i n e w a s o b s e r v e d , w i t h the e x c e p t i o n o f the T 1 9 0 P mutant ( T a b l e 3.4).  Tos-Gly-Pro-Arg-AMC  Tos-Gly-Pro-Lys-AMC  SR/SK*  kcat  kcat / K  (min")  (MM)  (min" uM")  (min-)  (MM)  (min" uM")  SGT  4880 ± 4 1 0  2.3 ± 0 . 2  2122  1670±120  3.6 ± 0.4  464  4.6  bSGT  4570±1210  2.0 ± 0 . 2  2285  1520 ± 6 0  3.2 ± 0 . 2  475  4.8  T190A  4950 ± 470  12.9 ± 1.0  384  192 ± 32  4.4 ± 0.6  44  8.7  T190P  3610±130  67 ± 5  54  527 ± 2  166 ± 2  3  18  T190S  6036 ± 561  6.1 ± 0 . 6  990  2584 ± 1 4 1  10.1 ± 1.2  256  3.9  T190V  2300 ± 1 7 7  224 ± 20  11  791 ± 13  231 ± 2  3.4  3.2  SR/SK = ( Tos-Gly-Pro-Arg-AMC  Table 3.3  kcat / K  kcat  m  kcat /  K )/(Tos-Gly-Pro-Lys-AMC r a  m  kcat / K  P I A r g i n i n e to L y s i n e preference o f mutant S G T e n z y m e s . A r g : L y s  preference w a s m e a s u r e d b y a m i d o l y t i c a c t i v i t y o f the native ( S G T ) , r e c o m b i n a n t ( b S G T ) a n d mutants o f S G T u s i n g t w o f l u o r o g e n i c peptides, T o s G l y - P r o - A r g - A M C a n d T o s - G l y - P r o - L y s - A M C . V a l u e s o b t a i n e d i n triplicate ± S.D.  47  r a  )  K; (uM) bSGT  2.7 ± 0 . 8  T190A  9.9 ± 0.7  T190P  16.4 ± 2 . 2  T190S  4.8 ± 0 . 8  T190V  197 ± 37  Table 3.4 K j values o f b e n z a m i d i n e f o r r e c o m b i n a n t S G T a n d the four mutant forms.  3.3.3. Crystallization and Structure Refinement X - r a y diffraction data were obtained f o r the T 1 9 0 P mutant o f S G T at 1.9 A r e s o l u t i o n . D a t a c o l l e c t i o n a n d refinement statistics are g i v e n i n T a b l e 3.5. A s w i t h the r e c o m b i n a n t w i l d type, the mutant c r y s t a l l i z e d i n the C222i space group, c o n t a i n e d o n e m o l e c u l e p e r a s y m m e t r i c unit a n d a M a t t h e w s c o e f f i c i e n t o f 2.3 A / D a . T h e mutant structure w a s deposited 3  i n the P D B database as 1 0 S 8 . D u r i n g refinement, l o w R yst a n d Rf cr  ree  values were obtained  (0.17 a n d 0.21). T h e s e were a c c o m p a n i e d b y e x c e l l e n t stereochemistry ( F i g u r e 3.1).  48  T190P SGT Data collection Resolution ( A )  1.9 (1.93 - 2 . 0 5 )  Total Observations  14471  C o m p l e t e n e s s (%)  89.7 (79.1)  Average redundancy  7.1  Vol  4 4 . 4 (25.2)  Rmerge (%)  3.1 (5.6)  R e f i n e m e n t statistics Space group  C222,  C e l l d i m e n s i o n s (a,b,c) *  5 0 . 0 8 , 6 9 . 6 0 , 119.83  M o l e c u l e s per a s y m m e t r i c u n i t  1  Rcryst  0.167  Rfree  0.212  Protein  atoiW  1632  S o l v e n t atoms per a s y m m e t r i c u n i t  193  A v e r a g e B - f a c t o r for p r o t e i n ( A )  12.8  A v e r a g e B - f a c t o r for water ( A )  21.4  Occupancy of C a  0.43 (17.50)  2  2  2 +  (B A ) 2  B o n d length deviations (A)  0.007  B o n d angle deviations (°)  1.4  * a = P = y = 9 0 ° ; i n c l u d i n g alternate side c h a i n c o n f o r m a t i o n s T  Table 3.5 D a t a  c o l l e c t i o n and refinement statistics for the T 1 9 0 P mutant o f  S G T i n c o m p l e x w i t h b e n z a m i d i n e . Statistics for the highest r e s o l u t i o n s h e l l are g i v e n i n parentheses.  49  Phi  (degrees)  F i g u r e 3.1 R a m a c h a n d r a n p l o t o f the crystal structure o f the T 1 9 0 P mutant o f S G T . A l l n o n - g l y c i n e residues are i n the a l l o w e d c o n f o r m a t i o n . T h e p l o t w a s c a l c u l a t e d u s i n g P R O C H E C K [110]  3.3.4 C a  2 +  b i n d i n g site o f B . s u b t i l i s d e r i v e d S G T  In the p r e v i o u s l y reported structure ( 1 S G T ) the c a l c i u m b i n d i n g site c o n s i s t e d o f the A s p 165 and G l u 2 3 0 c a r b o x y l a t e groups, two w e l l ordered water m o l e c u l e s a n d the c a r b o n y l o x y g e n atoms o f residues A l a l 7 7 a and G l u l 8 0 (Figure 3.2) [76]. In the present structures, A s p l 6 5 was s h o w n to adopt a different c o n f o r m a t i o n than o b s e r v e d p r e v i o u s l y . In the w i l d type r e c o m b i n a n t S G T structure, the carboxylate o f A s p 165 forms a bidentate electrostatic interaction w i t h A r g l 6 9 rather than the structural C a  2 +  i o n . In the T 1 9 0 P m o d e l , A s p l 6 5  adopts a p a i r o f alternative c o n f o r m a t i o n s , either f a c i n g the C a  50  2 +  i o n or towards A r g l 6 9 .  M o d e l i n g o f the t w o p o s i t i o n s at h a l f o c c u p a n c y generated a l o w e r B -factor for the c o n f o r m a t i o n i n v o l v e d i n the electrostatic interaction w i t h A r g l 6 9 . I n b o t h structures, the c a r b o n y l o x y g e n o f A l a l 7 7 a was oriented a w a y f r o m the C a  2 +  i o n and does not appear to be  i n v o l v e d i n the interaction. T h r e e ordered water m o l e c u l e s are associated w i t h the i o n , rather than the t w o p r e v i o u s l y observed. In contrast, a single d i s o r d e r e d water m o l e c u l e w i t h a h i g h B - f a c t o r ( 4 1 . 4 A ) was f o u n d near the C a 2  t r y p s i n - l i k e proteases, C a  2 +  2 +  i o n i n the T 1 9 0 P crystal structure. In other  i o n s have been f o u n d p r e v i o u s l y w i t h h i g h B - f a c t o r s relative to  the o v e r a l l structure suggesting less than f u l l o c c u p a n c y (25). T h e r e d u c e d o c c u p a n c y o f the ion is not s u r p r i s i n g as a related e n z y m e ,  Streptomyces erythraeus t r y p s i n , l a c k s G l u 2 3 0 and  n o c a l c i u m is evident i n the structure [121]. M o r e o v e r , the suggested C a  2 +  b i n d i n g site i n S G T  i s c o m p l e t e l y different than that o b s e r v e d i n m a m m a l i a n t r y p s i n - l i k e e n z y m e s [76]. W h e n taken w i t h p r e v i o u s observations that the c a l c i u m i o n p l a y s no r o l e i n catalysis but p l a y s a r o l e o n l y i n structural stability i n l o w / h i g h p H solutions, these data suggest that the c a l c i u m b i n d i n g site has w e a k affinity for the i o n [72].  3.3.5 T190S a n d the Loss of 7 - C H 3 D e g r a d a t i v e proteases i n v o l v e d i n digestive and protective functions t y p i c a l l y possess Ser or T h r residues at p o s i t i o n 190. H o w e v e r , proteases e x h i b i t i n g h i g h e r substrate s p e c i f i c i t y m a y also possess these residues [120]. T h e T 1 9 0 S mutant o f S G T demonstrated n o significant increase i n o v e r a l l substrate s p e c i f i c i t y , yet a m i n o r increase i n catalytic a c t i v i t y (kc at increase o f 2 5 % ) and a 3-fold increase i n K  m  ( T a b l e 3.3) were observed for b o t h the A r g and L y s  c o n t a i n i n g substrates. L o s s o f the y - C H is u n l i k e l y to s i g n i f i c a n t l y increase the solvent 3  a c c e s s i b i l i t y o f D 1 8 9 , and it is m o r e l i k e l y that the increased m o b i l i t y o f the y - O H results i n  51  the increased K  m  values for b o t h substrates. T h i s is v a l i d i f koai is used as a measure o f the  stabilization o f the transition state. In e u k a r y o t i c t r y p s i n - l i k e e n z y m e s that c o n t a i n Ser at p o s i t i o n 190, the space that w o u l d be o c c u p i e d b y a y - C H is f i l l e d b y m e t h y l g r o u p s f r o m 3  either residue 16 or 138 u s u a l l y i n the f o r m o f i s o l e u c i n e side c h a i n s . In S G T , both o f these residues are valine and possess one fewer m e t h y l group.  F i g u r e 3.2 C o m p a r i s o n o f the C a  2 +  b i n d i n g site i n S G T e n z y m e s : ( A ) native,  ( B ) w i l d - t y p e r e c o m b i n a n t and ( C ) T 1 9 0 P mutant o f S G T . T h e n u m b e r o f water m o l e c u l e s ( • ) that co-ordinate the structural c a l c i u m i o n (O),  as w e l l as  the c o n f o r m a t i o n o f the a m i n o a c i d ligands differ i n a l l three structures.  52  3.3.6 T190V and the Effect of a Branched Side Chain In contrast to the k i n e t i c s o f T 1 9 0 S , the T 1 9 0 V mutant demonstrated a 2 - f o l d reduction i n  k c  a  t  i n c o m b i n a t i o n w i t h nearly a 1 0 0 - f o l d increase i n K  m  for b o t h A r g and L y s  c o n t a i n i n g substrates ( T a b l e 3.3). R e p l a c e m e n t o f the y - O H w i t h a m e t h y l g r o u p r e m o v e s the h y d r o g e n b o n d i n g c a p a c i t y o f the residue and reduces the solvent a c c e s s i b i l i t y o f D 1 8 9 . T h i s results i n a d e s t a b i l i z e d transition state c o m p l e x relative to the w i l d - t y p e protease and a w e a k e r electrostatic interaction between the substrate and e n z y m e . T h e 1 0 0 - f o l d increase i n K  m  for A r g c o n t a i n i n g substrates c o m p a r e d to the 7 0 - f o l d increase i n K  m  for L y s c o n t a i n i n g  substrates indicates the s m a l l increase i n v o l u m e has a m o r e s i g n i f i c a n t effect o n the l o n g e r and b u l k i e r a r g i n y l side c h a i n . D 1 8 9 is not t y p i c a l l y observed w i t h V I 9 0 or 116 i n naturally o c c u r r i n g t r y p s i n - l i k e e n z y m e s l i k e l y due to the p o o r catalytic e f f i c i e n c y o f these c o m b i n a t i o n s o f side chains.  3.3.7 T190A and the Loss of y-OH A majority o f vertebrate e n z y m e s i n v o l v e d i n p h y s i o l o g i c a l r e g u l a t i o n , s u c h as the c o a g u l a t i o n factor serine proteases, possess A l a at p o s i t i o n 190 and e x h i b i t A r g : L y s substrate specificities r a n g i n g f r o m 7:1 for c o a g u l a t i o n factor X a to greater than 14:1 for b o v i n e t h r o m b i n [119]. T h e T 1 9 0 A m u t a t i o n i n S G T demonstrates the m o l e c u l a r basis for the p r e d o m i n a n c e o f A l a at this p o s i t i o n i n h i g h l y specific proteases f a v o r i n g a P I A r g residue. O p t i m a l rates o f catalysis at l o w concentrations o f substrate tend to be requisite characteristics o f these vertebrate e n z y m e s . K i n e t i c analysis o f the T 1 9 0 A mutant r e v e a l e d no change i n for A r g c o n t a i n i n g substrates but rather a 1 0 - f o l d r e d u c t i o n i n  k c  a  t  for L y s c o n t a i n i n g  substrates ( T a b l e 3.3). A s noted p r e v i o u s l y , the increase i n solvent a c c e s s i b i l i t y o f D 1 8 9  53  k c  a  t  s h o u l d stabilize the transition state c o m p l e x . H o w e v e r , the loss o f h y d r o g e n b o n d i n g ( p r e v i o u s l y p r o v i d e d b y the y - O H ) exhibits a m o r e p r o n o u n c e d effect o n the L y s c o n t a i n i n g substrate due to its r e q u i r e m e n t o f an ordered b r i d g i n g water m o l e c u l e to D 1 8 9 .  3.3.8 T190P U n l i k e the T 1 9 0 A m u t a t i o n , w h o s e effects w e r e p r e d o m i n a n t l y o n the k c o f the L y s at  c o n t a i n i n g substrate, the T 1 9 0 P m u t a t i o n affected the K  m  significandy. The K  m  for the A r g  c o n t a i n i n g substrate was 3 5 - f o l d h i g h e r than the w i l d - t y p e , c o m p a r e d to the 4 6 - f o l d increase for the L y s c o n t a i n i n g substrate (Table 3.3). S i m i l a r l y , the r e d u c t i o n i n k c for the A r g at  substrate (25%) was s i g n i f i c a n t l y less than the L y s substrate ( 6 6 % ) . Together, these changes generate an o v e r a l l A r g to L y s preference o f 18 to 1. A p r e v i o u s report [66] a n a l y z e d the S 1 9 0 P mutant o f rat a n i o n i c t r y p s i n , w h i c h is analogous to the T 1 9 0 P constructed i n S G T . W h e n u s i n g the same p a i r o f substrates used i n this study, w i l d - t y p e rat a n i o n i c t r y p s i n e x h i b i t s a s i m i l a r p r i m a r y substrate s p e c i f i c i t y to S G T . H o w e v e r , the S 1 9 0 P m u t a t i o n i n rat a n i o n i c t r y p s i n results i n a h i g h l y specific protease that favors the A r g substrate 1 3 5 - f o l d over the L y s c o n t a i n i n g substrate. T h i s increase i n substrate s p e c i f i c i t y was c o m b i n e d w i t h a greater than 1 0 - f o l d r e d u c t i o n i n k c for b o t h A r g and L y s substrates. T h e s e authors suggested at  that T y r 2 2 8 m a y be i n v o l v e d i n steric c l a s h i n g w i t h the p r o l i n e r i n g at p o s i t i o n 190, l e a d i n g to r e d u c e d activity. In S G T , residue 2 2 8 is also T y r suggesting an alternate b i n d i n g m o d e o f this mutant. T o address these discrepancies, the c r y s t a l structure o f the T 1 9 0 P mutant i n c o m p l e x w i t h the s m a l l m o l e c u l e i n h i b i t o r b e n z a m i d i n e was investigated. B i n d i n g o f the b e n z a m i d i n e i n h i b i t o r to the T 1 9 0 P mutant is nearly i d e n t i c a l to that o b s e r v e d i n other t r y p s i n - l i k e proteases [112]. T h e p r o l i n e r i n g o f residue 190 does not adopt  54  a c o n f o r m a t i o n that o c c l u d e s the n e g a t i v e l y c h a r g e d c a r b o x y l a t e g r o u p o f A s p 189, n o r does it c o n f l i c t w i t h T y r 2 2 8 suggesting that the p r e v i o u s l y characterized s p e c i f i c i t y o f the S 1 9 0 P mutant i n rat a n i o n i c t r y p s i n was the result o f s e c o n d s h e l l residues at p o s i t i o n s 16 or 138 ( F i g u r e 3.3). T h e m u t a t i o n does not s i g n i f i c a n t l y affect the c o n f o r m a t i o n o f any o f the residues s u r r o u n d i n g T 1 9 0 P , i n c l u d i n g the c r i t i c a l A s p l 8 9 . T h e l o c a l r.m.s. d e v i a t i o n is l o w (0.70 A) for a l l 9 6 atoms w i t h i n a 5 A radius o f residue 190. H o w e v e r , the b a c k b o n e c a r b o n y l g r o u p o f A s p l 8 9 is rotated 4 5 ° relative to the w i l d - t y p e structure due steric constraints o f the p r o l i n e residue. R o t a t i o n o f this c a r b o n y l g r o u p does not disrupt the h y d r o g e n b o n d w i t h the b a c k b o n e n i t r o g e n o f residue 17. H e n c e , the moderate increase i n A r g to L y s substrate s p e c i f i c i t y o f this mutant is the l i k e l y the result o f the strengthened interaction b e t w e e n the substrate and A s p 189 i n a m o r e h y d r o p h o b i c e n v i r o n m e n t . T h e effect o n l y s y l - s i d e c h a i n s is m o r e p r e d o m i n a n t due to the l a c k o f h y d r o g e n b o n d i n g o f the p r o l i n e r i n g to the substrate or a b r i d g i n g water m o l e c u l e and w o u l d reduce the rate o f association o f the side c h a i n w i t h Aspl89.  55  F i g u r e 3.3 C o m p a r i s o n o f the S I b i n d i n g pocket i n S G T e n z y m e s : ( A ) the r e c o m b i n a n t w i l d - t y p e structure and ( B ) the T 1 9 0 P mutant o f S G T c o m p l e x e d w i t h the b e n z a m i d i n e i n h i b i t o r ( B e n z ) . In the w i l d - t y p e structure, the y - O H points towards the S1 p o c k e t and p r o v i d e s a H - b o n d i n g g r o u p for the substrate. M u t a t i o n o f residue 190 to p r o l i n e removes this H - b o n d i n g c a p a c i t y w i t h o u t d i s r u p t i n g the c r i t i c a l D 1 8 9 .  A l t h o u g h A s p 189 adopts a s i m i l a r c o n f o r m a t i o n to that f o u n d i n the w i l d - t y p e protease, it is p o s s i b l e that the b i n d i n g o f the b e n z a m i d i n e i n h i b i t o r stabilizes the c o n f o r m a t i o n o f this side c h a i n through f o r m a t i o n o f the electrostatic interaction. A n a l y s i s o f  56  the i n h i b i t i o n constants o f b e n z a m i d i n e w i t h the w i l d - t y p e r e c o m b i n a n t protease and four mutants reveals the basis for the i n c r e a s e d s p e c i f i c i t y w i t h o u t loss o f catalytic a c t i v i t y . S i m i l a r f o l d differences for the K  m  values for the peptide substrates and  values relative to  the w i l d - t y p e are observed for a l l mutants except T 1 9 0 P . T h e Kj v a l u e is s i x - f o l d h i g h e r than the w i l d - t y p e , whereas the K  m  values for the A r g and L y s c o n t a i n i n g substrates increase 3 5 -  f o l d and 4 6 - f o l d , r e s p e c t i v e l y (Tables 3.3 & 3.4). A s the i n h i b i t o r forms a direct electrostatic interaction w i t h A s p 189, the m i n o r difference o f the i n h i b i t o r y constants suggests that i n the T 1 9 0 P mutant the interaction occurs i n a m o r e h y d r o p h o b i c e n v i r o n m e n t and is not a c c o m p a n i e d b y structural rearrangement o f the S 1 b i n d i n g p o c k e t . W h e r e a s T 1 9 0 A demonstrates the interactions present at the S I b i n d i n g p o c k e t i n the majority o f proteases w i t h a P I A r g preference, the k i n e t i c analysis o f T 1 9 0 P suggests a potential intermediate i n the e v o l u t i o n o f the vertebrate c o a g u l a t i o n cascade. O n l y t w o natural proteases have been i d e n t i f i e d that possess A s p 189 and P r o 190 - h a g f i s h p r o t h r o m b i n and h u m a n k a l l i k r e i n 10, yet neither p r o t e i n has been characterized w i t h respect to substrate specificity. B o t h genes have been characterized b y D N A sequence analysis o f a n u m b e r o f o v e r l a p p i n g c D N A l i b r a r y c l o n e s [122-124]. T h e presence o f P r o at this p o s i t i o n suggests a s i m i l a r s p e c i f i c i t y as that o b s e r v e d for the T 1 9 0 P o f S G T . M o r e o v e r , the residues s u r r o u n d i n g residue 190 are i d e n t i c a l to those f o u n d i n S G T . D N A sequence analysis o f the h a g f i s h p r o t h r o m b i n gene reveals that m a n y o f the features attributed to substrate s p e c i f i c i t y , s u c h as the 6 0 - and 9 9 - l o o p s , are i d e n t i c a l . H e n c e , the r e d u c e d catalytic a c t i v i t y and h i g h e r K  m  values  c a u s e d b y the p r o l i n e at this p o s i t i o n m a y be c o m p e n s a t e d b y an increased c o n c e n t r a t i o n o f the e n z y m e or substrate i n the b l o o d stream o f this p r i m i t i v e vertebrate.  57  3.3.9 Second Shell Residues A n u m b e r o f catalytic or structural studies i n v o l v i n g the structure based d e s i g n o f e n z y m e properties have demonstrated an important r o l e for s e c o n d s h e l l residues s u r r o u n d i n g the m u t a t i o n o f interest [125]. In serine proteases, residue 190 interacts d i r e c t l y w i t h the side chains o f residues 16 (the N - t e r m i n u s o f the protein), 138 and 228. R e s i d u e 228 is a h i g h l y c o n s e r v e d tyrosine i n k n o w n t r y p s i n - l i k e e n z y m e s . R e s i d u e s 16 and 138 are restricted to h y d r o p h o b i c side c h a i n s ( V a l , He, and L e u ) . T h e differences i n k i n e t i c parameters o b s e r v e d b e t w e e n mutations made i n this study, rat a n i o n i c t r y p s i n and h u m a n t r y p s i n (type I) are l i k e l y due to the presence or absence o f m e t h y l groups w i t h i n this p a i r o f residues [66,67,119]. M o r e o v e r , mutagenesis o f residue 16 i n rat a n i o n i c t r y p s i n o g e n II has been demonstrated to affect p r i m a r y substrate s p e c i f i c i t y o f the e n z y m e [126]. A m o r e detailed i n v e s t i g a t i o n o f these residues is r e q u i r e d to understand the basis o f substrate s p e c i f i c i t y w i t h i n this f a m i l y o f important enzymes.  3.4 Conclusions O n the basis o f the ease o f p r o d u c t i o n o f S G T i n the  B.  subtilis e x p r e s s i o n  system, it is  p o s s i b l e to enhance the s p e c i f i c i t y at the S 2 to S 4 b i n d i n g pockets b y either structure-based d e s i g n or a directed e v o l u t i o n strategy. I n t r o d u c t i o n o f an affinity tag, s u c h as a h e x a h i s t i d i n e tag, w o u l d speed the p u r i f i c a t i o n process o f the p r o t e i n and facilitate characterization o f r e c o m b i n a n t mutant proteases. T h e a b i l i t y to c r y s t a l l i z e this m o l e c u l e r e a d i l y supports S G T as a m o d e l scaffold for understanding the m e c h a n i s m s o f substrate s p e c i f i c i t y i n the h i g h l y e v o l v e d serine protease f a m i l y .  58  Chapter 4. Engineering Coagulation Factor X a Substrate Specificity into Streptomyces griseus Trypsin  4.1 Introduction 4.1.1 Overview A r c h i t e c t u r e o f the active site p l a y s a k e y role i n the p h y s i o l o g i c a l functions o f serine proteases. A l t h o u g h the catalytic m a c h i n e r y is s i m i l a r , i f not i d e n t i c a l , w i t h i n the f a m i l y o f serine proteases, the residues c o m p r i s i n g the active site dictate f u n c t i o n . D i f f e r e n c e s i n the active site l e a d to substrate s p e c i f i c i t y and the l e v e l o f r e g u l a t i o n b y protease i n h i b i t o r s . D e t a i l e d understanding o f the m o l e c u l a r basis o f substrate s p e c i f i c i t y is needed to d e s i g n i n h i b i t o r s for therapeutic applications. M o r e o v e r , the a b i l i t y to t a i l o r protease s p e c i f i c i t y to meet s p e c i a l i z e d needs is an attainable g o a l . A t present, it has not been established what m a x i m u m l e v e l s o f substrate s p e c i f i c i t y c o u l d e x i s t o n the serine protease scaffold. F e w studies have i n v o l v e d i m p r o v i n g the extended substrate s p e c i f i c i t y o f serine proteases. Proteases have been e x t e n s i v e l y characterized w i t h respect to e n z y m a t i c properties. Rates o f catalysis against numerous libraries o f peptide and p o l y p e p t i d e substrates that are b o t h natural and synthetic i n o r i g i n are r e a d i l y a v a i l a b l e [41,127-131]. I n h i b i t i o n constants are s i m i l a r l y abundant due to the i m p o r t a n c e o f p h y s i o l o g i c a l r e g u l a t i o n and for i m p e d i n g the p r o g r e s s i o n o f a p a t h o l o g y or p r e v e n t i n g one f r o m d e v e l o p i n g [132-137]. I n a d d i t i o n , a vast amount o f sequence and structural data c a n be f o u n d i n the databases. Indeed, the quantity o f i n f o r m a t i o n c o m p i l e d o n proteases is d a u n t i n g . U l t i m a t e l y these data reflect properties that  59  result f r o m a p p r o x i m a t e l y fifty a m i n o acids that create and surround the active site o f serine proteases. L e s s than twenty a m i n o a c i d residues are i n v o l v e d i n enzyme-substrate interactions i n the S I peptidases [24]. A n u m b e r o f residues i n v o l v e d i n substrate b i n d i n g exert their i n f l u e n c e v i a b a c k b o n e contacts or b y s t a b i l i z a t i o n o f the entire structure o f the protease d o m a i n . S o m e e x a m p l e s i n c l u d e residues 2 1 4 to 2 1 6 w h i c h are i n v o l v e d i n f o r m a t i o n o f the anti-parallel P-strand b e t w e e n e n z y m e and substrate and three d i s u l f i d e bonds b e t w e e n residues 4 2 and 5 8 , 168 and 182, and 191 and 2 2 0 that stabilize the entire d o m a i n . In both instances, these residues are h i g h l y c o n s e r v e d throughout the serine protease f a m i l y and d o not s i g n i f i c a n t l y m o d u l a t e specificity, but rather assist i n the enzyme-substrate interactions and stabilize the transition state o f the catalytic process [138,139]. T h u s , v a r i a t i o n i n substrate specificity c a n be r e d u c e d i n c o m p l e x i t y to r o u g h l y ten a m i n o a c i d p o s i t i o n s . T h e s e residues are located o n a l l sides o f the active site and their effects c a n be altered b y their l o c a l e n v i r o n m e n t s and p r o x i m a l residues. E x t e n d e d substrate specificity is a h a l l m a r k o f the serine proteases o f vertebrate b l o o d c o a g u l a t i o n . T h e s e proteases serve as useful m o d e l s to understand substrate s p e c i f i c i t y throughout the entire f a m i l y o f S I peptidases. C o a g u l a t i o n factor X a ( F X a ) is i m p o r t a n t due to its central r o l e i n c o a g u l a t i o n and w i d e spread use i n b i o t e c h n o l o g y - r e l a t e d a p p l i c a t i o n s r e q u i r i n g site specific p r o t e o l y s i s . T h e p r e d o m i n a n t feature o f F X a substrate specificity is a t w o residue l o o p at p o s i t i o n 99 [140]. Insertion at this site i n the sequence p o s i t i o n s a large aromatic side c h a i n , T y r 9 9 , i n the S 2 p o c k e t and restricts P 2 substrate residues to s m a l l G l y side c h a i n s . L e s s is k n o w n about the other determinants o f substrate specificity i n F X a .  60  K i n e t i c data for the h y d r o l y s i s o f a n u m b e r o f different peptide substrates suggests that the S 3 and S 4 pockets o f F X a d i s p l a y m i n i m a l s e l e c t i v i t y [41]. T h e S 3 s p e c i f i c i t y p o c k e t is t y p i c a l l y the least stringent o f the p o c k e t s throughout the S 1 peptidase f a m i l y due to the solvent exposure o f the P 3 side c h a i n . M o d e r a t e l y s i z e d h y d r o p h o b i c a m i n o acids are preferred b y the S 4 b i n d i n g p o c k e t w h i c h is d o m i n a t e d b y h y d r o p h o b i c side chains [116]. G i v e n the s e l e c t i v i t y o f these pockets, it seems p o s s i b l e to engineer F X a - l i k e s p e c i f i c i t y into a b r o a d l y specific protease. M o r e o v e r , it is l i k e l y that a protease w i t h a stricter preference for the I l e - G l u - G l y - A r g F X a cleavage sequence c o u l d be engineered. M e a s u r e s o f substrate s p e c i f i c i t y o f a protease c a n differ substantially based o n the type o f substrate e m p l o y e d . P r o t e o l y t i c cleavage o f a short peptide c a n misrepresent the rate o f h y d r o l y s i s o f a single b o n d i n a f o l d e d protein. D e t a i l e d characterization o f F X a u s i n g c o m p r e h e n s i v e peptide substrate libraries revealed that large, planar h y d r o p h o b i c residues were preferred i n the S 2 p o c k e t over s m a l l residues [41,42]. S u c h an o b s e r v a t i o n contrasts w i t h sequence analysis o f predominant  in vivo F X a substrates. A m i n o  in vivo substrate o f F X a ,  a c i d sequences o f p r o t h r o m b i n , the  f r o m organisms s p a n n i n g 4 5 0 m i l l i o n years o f  vertebrate e v o l u t i o n s h o w c o n s e r v a t i o n o f the I l e - G l u / A s p - G l y - A r g r e c o g n i t i o n sequence [122,123]. M o r e o v e r , n u m e r o u s studies have e m p l o y e d F X a for the site specific p r o t e o l y s i s o f r e c o m b i n a n t proteins [127]. R e p o r t s o f n o n - s p e c i f i c proteolysis b y F X a are o b v i o u s l y not easy to f i n d i n the literature. H o w e v e r , the large number o f successful results c o n f i r m s selectivity for the c l e a v a g e sequence is h i g h .  61  4.1.2 Choice of Mutations to Mimic FXa-like Specificity P r e v i o u s l y , I d e s c r i b e d the d e s i g n o f a protease w i t h a h i g h p r i m a r y s p e c i f i c i t y for A r g i n the P I p o s i t i o n u s i n g S G T . U s i n g the o p t i m a l mutant o f S G T f r o m this w o r k as a starting point, the d e s i g n o f F X a - l i k e s p e c i f i c i t y was pursued. T o c o n v e r t the s p e c i f i c i t y o f S G T into F X a , s i x mutations were i n t r o d u c e d into S G T to enhance s e l e c t i v i t y o f the S 2 , S 3 and S 4 pockets (Table 4.1). M u t a t i o n s were c h o s e n based on sequence c o n s e r v a t i o n i n k n o w n F X a proteins f r o m v a r i o u s species and i n s p e c t i o n o f various x - r a y c r y s t a l structures o f m e m b e r s o f the S I peptidase f a m i l y . T h e s e mutations i n S G T are i n v o l v e d i n enzyme-substrate interactions as w e l l as o p t i m i z a t i o n o f the o v e r a l l architecture o f the active site ( F i g u r e 4.1). M a n y o f the mutations created also m i m i c w h a t is f o u n d i n activated p r o t e i n C ( a P C ) , factor I X a ( F L X a ) , factor X I a , and factor V i l a ( F V I I a ) .  Name  Mutations  T190P  T190P  LP  99-loop, T 1 9 0 P  YP  99-loop, T 9 9 Y , T 1 9 0 P  YFP  99-loop, T 9 9 Y , N 1 7 4 F , T 1 9 0 P  YSFP  99-loop, T 9 9 Y , Y 1 7 2 S , N 1 7 4 F , T 1 9 0 P  YSFMP  99-loop, T 9 9 Y , Y 1 7 2 S , N 1 7 4 F , E 1 8 0 M , T 1 9 0 P  YSFMPE  99-loop, T 9 9 Y , Y 1 7 2 S , N 1 7 4 F , E 1 8 0 M , T 1 9 0 P , Y217E  Table 4.1  M u t a n t s o f S G T constructed to m i m i c the substrate s p e c i f i c i t y o f F X a . F o r  the 9 9 - l o o p mutation, t w o residues ( L y s and G l u at p o s i t i o n s 96 and 97) were inserted s i m i l a r to that f o u n d i n the F X a p o l y p e p t i d e sequence.  62  S e l e c t i v i t y o f the S 2 p o c k e t i n F X a is generated b y the i n s e r t i o n o f t w o a m i n o a c i d residues at p o s i t i o n 9 9 . E x t e n s i o n o f the p o l y p e p t i d e c h a i n at this l o c a t i o n p o s i t i o n s the T y r 9 9 side c h a i n into the S 2 p o c k e t and restricts access to s m a l l aliphatic side chains [140]. T w o mutations i n S G T were r e q u i r e d to engineer selectivity for P 2 side chains. F i r s t , a t w o residue insertion was created. S e c o n d , T h r 9 9 T y r was added to the l o o p construct to c o n s t r a i n the S 2 p o c k e t i n a s i m i l a r fashion to F X a .  Figure 4.1  R e s i d u e s i n v o l v e d i n the extended substrate s p e c i f i c i t y o f c o a g u l a t i o n  proteases. T h e N a b i n d i n g site is k n o w n to p l a y a r o l e i n the substrate s p e c i f i c i t y o f +  t h r o m b i n and lies adjacent to the S I pocket.  E 1 8 0 M w a s i n t r o d u c e d into S G T to r e m o v e the electrostatic effects o f the n e g a t i v e l y c h a r g e d G l u residue and m i m i c a residue c o n s e r v e d i n the entire S I f a m i l y peptidases. Sequence analysis s h o w s that M e t 180 is h i g h l y c o n s e r v e d and exists i n a p p r o x i m a t e l y 6 0 % o f the f a m i l y . H o w e v e r , a f u n c t i o n a l role for residue 180 has not been d e s c r i b e d i n the literature. R e s i d u e G l u 180 i n S G T is one o f several n e g a t i v e l y c h a r g e d side chains near the S 4 p o c k e t  63  that generates an o v e r a l l negative electrostatic potential i n the r e g i o n . A s h y d r o p h o b i c P 4 residues are preferred i n F X a , it was conjectured that r e m o v a l o f the c h a r g e d m o i e t y i n the S 4 p o c k e t w o u l d i m p r o v e substrate b i n d i n g . Y 1 7 2 S and N 1 7 4 F mutations i n S G T were constructed to facilitate p r o p e r p o s i t i o n i n g o f the 1 7 2 - l o o p and increase the h y d r o p h o b i c i t y o f the S 4 p o c k e t . In S G T , T y r l 7 2 is b u r i e d i n the core o f the structure i n a s i m i l a r f a s h i o n to other t r y p s i n - l i k e e n z y m e s [76]. H o w e v e r , Ser 172 i n F X a has a c o n f o r m a t i o n that exposes the side c h a i n to solvent and is i n v o l v e d i n the p o s i t i o n i n g o f residue 174 [141]. M u t a t i o n at the equivalent p o s i t i o n s i n b o v i n e t r y p s i n l e d to a f l e x i b l e 1 7 2 - l o o p and suggests replacement o f residues 172 to 174 is not sufficient to create the S 4 p o c k e t o f F X a [142]. T h u s , s t a b i l i z a t i o n o f the l o o p s h o u l d be important f o r o p t i m i z a t i o n o f the extended substrate s p e c i f i c i t y i n F X a . Y 2 1 7 E was created i n S G T to secure the 1 7 2 - l o o p c o n f o r m a t i o n o b s e r v e d i n F X a . R e s i d u e 217 is p o o r l y c o n s e r v e d i n the S I f a m i l y , yet c o n s e r v e d i n a l l c o a g u l a t i o n proteins i d e n t i f i e d f r o m a variety o f vertebrate species [122,123]. In the c r y s t a l structure o f h u m a n F X a , residue 2 1 7 forms t w o charge assisted h y d r o g e n bonds w i t h S e r l 7 3 v i a the h y d r o x y l g r o u p o f the side c h a i n and a m i d e g r o u p o f the b a c k b o n e [143]. U n f o r t u n a t e l y , the G l y l 7 3 S e r mutant o f S G T has not been c h a r a c t e r i z e d yet. Using  B. subtilis as  an e x p r e s s i o n host, a n u m b e r o f mutants at the residues d i s c u s s e d  above were constructed i n S G T and their e n z y m a t i c properties determined. Substrate s p e c i f i c i t y was investigated b y c o m p a r i s o n o f the rates o f h y d r o l y s i s o f a s m a l l l i b r a r y o f c o m m e r c i a l l y a v a i l a b l e c h r o m o g e n i c peptides. T h e mutant b e a r i n g a l l seven mutations d i d not p r o d u c e a protease w i t h F X a - l i k e specificity, but rather s p e c i f i c i t y towards c o a g u l a t i o n factor X I a ( F X I a ) substrates was o b s e r v e d . A n intermediate mutant w i t h s i x mutations l e d to a  64  protease w i t h moderate F X a - l i k e specificity. T h e s e results c o n f i r m p r e v i o u s studies that demonstrated a r o l e for the 9 9 - l o o p a n d 1 7 2 - l o o p i n F X a and related e n z y m e s . R e s i d u e 2 1 7 is c o n f i r m e d as a determinant o f substrate specificity. H o w e v e r , questions about the detailed r o l e o f residue 217 are raised b y the data. Further, residue 180 has been i d e n t i f i e d as a determinant o f the substrate s p e c i f i c i t y i n the S I f a m i l y o f peptidases.  4.2 Materials & Methods 4.2.1 Plasmids, Bacterial Strains, and Growth Conditions E. coli w a s g r o w n u s i n g standard methods [90]. P l a s m i d D N A was p u r i f i e d u s i n g a Q I A p r e p s p i n m i n i p r e p k i t ( Q i a g e n ) a n d m a n i p u l a t e d u s i n g standard p r o t o c o l s [90]. E n z y m e s w e r e obtained f r o m N e w E n g l a n d B i o l a b s a n d R o c h e M o l e c u l a r B i o c h e m i c a l s .  B. subtilis  strain W B 7 0 0 w a s g r o w n i n super-rich m e d i u m [91] o r o n tryptose b l o o d agar base ( D i f c o ) at 3 7 ° C . F o r the  B. subtilis c a r r y i n g p l a s m i d p W B 9 8 0 [92], k a n a m y c i n was added to a f i n a l  c o n c e n t r a t i o n o f 10 pig raL" i n b o t h l i q u i d a n d s o l i d m e d i a . 1  4.2.2 Construction of a Hexahistidine-tagged SGT P l a s m i d S G T p E T - 2 8 ( a ) + w a s p r e v i o u s l y constructed i n an u n s u c c e s s f u l attempt to express r e c o m b i n a n t S G T b y c l o n i n g the gene i n t o the X b a l a n d X h o l restriction endonuclease c l e a v a g e sites o f the p l a s m i d . I n s p e c t i o n o f electrospray mass spectrometry data o f aged samples o f r e c o m b i n a n t S G T f r o m  B. subtilis suggested that A r g 2 4 3 w a s w e a k l y  susceptible to autolysis. T h e m u t a t i o n A r g 2 4 3 S e r w a s constructed u s i n g the Q u i k C h a n g e sitedirected mutagenesis k i t (Stratagene) as suggested b y the manufacturer u s i n g the o l i g o n u c l e o t i d e 5 ' - G C C T C G G C C G C C A G C A C G C T C G A G C A C - 3 ' a n d reverse c o m p l e m e n t  65  o l i g o n u c l e o t i d e . T h e C - t e r m i n a l p o r t i o n o f the S G T gene was s u b - c l o n e d f r o m p E T 2 8 ( a ) + into b S G T p W B 9 8 0 v i a P v u I I a n d A v r l l cleavage sites ( F i g u r e 4.2). T h u s , S G T was c l o n e d i n frame w i t h a hexa-histidine tag ( H i s R p W B 9 8 0 ) . A s this p l a s m i d construct w a s to be used for  E. coli p l a s m i d ,  s u b - c l o n i n g mutants o f S G T f r o m the p B l u e s c r i p t K S +  a s m a l l p o r t i o n o f the  gene w a s deleted b y d i g e s t i n g w i t h N a r l and self-ligation to y i e l d A N a r l H i s R p W B 9 8 0 . A s a result, successful s u b - c l o n i n g o f mutants w o u l d restore protease a c t i v i t y .  Xbal  Xhol  +c  Xbal Xhol Avrll 1 KHHOHH^  ^  pET-28(a)+  Xbal  Xhol  Avrll  SGTTTS^S  +  Arg243Ser  Hindll I Xbal  A v r  U  H  bSGT pWB980  Hindlll Xhol HisR pWB980  ^ )  Hindlll Xhol Narl ANarl HisR pWB980  F i g u r e 4.2 P l a s m i d construction for the p r o d u c t i o n o f r e c o m b i n a n t H i s - t a g g e d S G T ( H i s R p W B 9 8 0 ) and a deletion mutant construct for e a s i l y i d e n t i f y i n g successful subc l o n i n g o f mutant S G T genes ( A N a r l H i s R p W B 9 8 0 ) .  66  4.2.3 Sequence analysis of the SI Family peptidases A l l sequences for the S I f a m i l y s u b - f a m i l y A peptidases were d o w n l o a d e d f r o m the M E R O P S database (http://www.merops.ac.uk, release date 0 4 - 0 3 - 2 0 0 2 ) [22]. Sequences were p r e - a l i g n e d b y the curators o f the database. T h e c o m p l e t e a l i g n m e n t o f 7 4 0 proteases w a s pasted into M i c r o s o f t E x c e l , and then c o n v e r t e d into s i n g l e c o l u m n s c o r r e s p o n d i n g to i n d i v i d u a l residues i n the p o l y p e p t i d e c h a i n . D i s t r i b u t i o n o f a m i n o acids at each p o s i t i o n o f interest was obtained u s i n g the C O U N T I F f u n c t i o n w i t h i n the p r o g r a m . T h e file served as a useful database that l i n k e d a m i n o acids at a d e f i n e d p o s i t i o n i n the p o l y p e p t i d e sequence to characterized e n z y m e s . R e g i o n s w h e r e insertions or deletions o c c u r r e d i n the f a m i l y were not h a n d l e d w e l l b y this m e t h o d and i n s p e c t i o n o f k n o w n c r y s t a l structures and p u b l i s h e d literature were r e q u i r e d .  4.2.4 DNA Manipulation U s i n g the p r e v i o u s l y d e s c r i b e d S G T gene c l o n e d into p B l u e s c r i p t K S + p l a s m i d ( C h p t . 2.2.2), mutagenesis was p e r f o r m e d o n the gene u s i n g a Q u i k C h a n g e site-directed mutagenesis k i t (Stratagene) as d e s c r i b e d b y the manufacturer. O l i g o n u c l e o t i d e s used for mutagenesis are p r o v i d e d i n T a b l e 4.2. D N A sequence analysis o f the c l o n e d gene and mutants was p e r f o r m e d u s i n g the B i g D y e T e r m i n a t o r k i t and a n a l y z e d o n a n A B I 3 7 0 0 D N A Sequencer ( A p p l i e d B i o s y s t e m s ) . M u t a n t S G T genes were s u b - c l o n e d into p l a s m i d A N a r l H i s R p W B 9 8 0 v i a H i n d l l l and P v u I I restriction sites and transformed into S p i z i z e n [93].  67  B. subtilis W B 7 0 0  b y the m e t h o d o f  Mutation  Oligonucleotide  99-Loop  5' - C A G G C C C C C C G G C T A C A A C A A G G A G G G C A C C G G C A A G G A C T G G - 3 '  T99Y  5' - C A A G G A G G G C T A C G G C A A G G A C - 3 '  E180M  5' - C T C G T G G C C A A C G  N174F  5' - T C C G C G T  Y172S  5'  AGATGATCTGCGCCGGATAC-3'  ACGGCTTCDGAGCTCGTGGCC-3'  -GCCGCTCCGCGTCCGGCTTCGAGCT-3'  N174F Y217E  5 ' -A G C T G G G G C G A G G G C T G C G C C - 3 '  Table 4.2 O l i g o n u c l e o t i d e s used to mutate the S G T gene to m i m i c residues f o u n d i n F X a . T h e reverse c o m p l e m e n t sequences o f these o l i g o n u c l e o t i d e s were also used i n the mutagenesis.  4.2.5 Purification of His-tagged SGT and Mutants thereof Overnight  B. subtilis 2 0 m L cultures  were used to inoculate 2 5 0 m L o f super-rich  broth c o n t a i n i n g 10 [ i g / m L k a n a m y c i n and g r o w n for 16 hrs at 3 7 ° C . T h e supernatant was harvested b y centrifugation (30 m i n . , 5 0 0 0 r p m ) and then passed o v e r a T a l o n affinity r e s i n c o l u m n ( B D B i o s c i e n c e s ) (10 c m x 0.75 c m ) e q u i l i b r a t e d i n w a s h buffer (50 m M s o d i u m phosphate, 5 0 0 m M N a C I , p H 8.2). T h e c o l u m n was w a s h e d w i t h 10 c o l u m n v o l u m e s o f w a s h buffer and the r e c o m b i n a n t p r o t e i n eluted w i t h w a s h buffer c o n t a i n i n g 100 m M i m i d a z o l e . A c t i v e fractions c o n t a i n i n g r e c o m b i n a n t p r o t e i n were concentrated u s i n g a 10,000 N M W L Ultrafree-4 centrifugal filter unit ( M i l l i p o r e ) and d i a l y z e d w i t h 100 m M T r i s - H C I , 150 m M N a C I , 2 0 m M  CaCl2, p H 7.6 i n the same unit. R e c o m b i n a n t proteins were stable at  4 ° C for m o n t h s . P r o t e i n quantification was i d e n t i c a l to that d e s c r i b e d p r e v i o u s l y ( C h p t . 2.2.3).  68  4.2.6 Characterization of Substrate Specificity K i n e t i c analysis was p e r f o r m e d i n 10 m M T r i s - H C I buffer c o n t a i n i n g 150 m M N a C I , 2 0 m M C a C l , and 0.1 % P E G 8 0 0 0 , p H 7.6 at 2 5 ° C . R e a c t i o n s (300 uT) were prepared i n 9 6 2  w e l l m i c r o p l a t e s u s i n g either a R o b o S e q 4 2 0 4 or R o b o G o laboratory a u t o m a t i o n s y s t e m ( M W G B i o t e c h A G , E b e r s b u r g , G e r m a n y ) and m e a s u r e d u s i n g a L a b s y s t e m s M u l t i s k a n A s c e n t plate reader. P e p t i d e substrates were h a n d l e d as suggested b y their manufacturers ( D i a p h a r m a , A m e r i c a n D i a g n o s t i c a ) . A s s e s s m e n t o f s p e c i f i c i t y u s i n g c h r o m o g e n i c peptide substrates was estimated b y direct analysis o f the h y d r o l y s i s o f each substrate at 4 0 \iM at t w o stages o f the p u r i f i c a t i o n . In this m e t h o d , the s p e c i f i c i t y constant (kc /Km) was determined at  f r o m the slope o f the natural l o g a r i t h m o f substrate r e m a i n i n g as f u n c t i o n o f time. D e t a i l e d k i n e t i c analyses were p e r f o r m e d o n a m i n i m u m o f s i x substrate concentrations r a n g i n g f r o m 2 0 to 6 0 0 p M and e n z y m e concentrations o f 10 to 7 0 n M . H i g h e r substrates concentration were not e x a m i n e d due to s o l u b i l i t y d i f f i c u l t i e s w i t h the peptides.  4.2.7 Macromolecular Substrate Specificity H u m a n p r o t h r o m b i n and F X a were p u r c h a s e d f r o m H a e m a t o l o g i c a l T e c h n o l o g i e s . P r o t h r o m b i n (3.5 p g ) was digested w i t h mutants o f S G T (60 ng) and F X a (60 ng) o v e r n i g h t at r o o m temperature i n 10 m M T r i s - H C I buffer c o n t a i n i n g 150 m M N a C I , 2 0 m M C a C b , and 0.1 % P E G 8 0 0 0 , p H 7.6. P r o t e o l y t i c fragments were r e s o l v e d b y S D S - P A G E f o l l o w i n g a standard p r o t o c o l [90].  69  4.3 Results & Discussion 4.3.1 Production of His-tagged S G T In order to facilitate p u r i f i c a t i o n and analysis o f a large n u m b e r o f mutants, s i m p l i f i c a t i o n o f the four step p u r i f i c a t i o n scheme d e s c r i b e d p r e v i o u s l y was r e q u i r e d ( C h p t . 2.2.3). Initial attempts to p r o d u c e r e c o m b i n a n t S G T i n  E. coli were unsuccessful,  yet  p r o d u c e d a construct b e a r i n g S G T i n frame w i t h a h e x a - h i s t i d i n e tag i n the p E T - 2 8 a ( + ) p l a s m i d . S e v e r a l h i s t i d i n e residues i n s u c c e s s i o n p r o m o t e b i n d i n g to metal i o n s s u c h as C u +2 Ni  + 2  ,  +2  , or C o  . A s the metal i o n s c a n be i m m o b i l i z e d onto an appropriate c h r o m a t o g r a p h y  m e d i u m , capture o f r e c o m b i n a n t p r o t e i n f r o m c o m p l e x samples is greatly s i m p l i f i e d . T h e C t e r m i n a l r e g i o n o f S G T b e a r i n g the tag was amenable for s u b c l o n i n g f r o m p E T - 2 8 a ( + ) into the B.  subtilis p l a s m i d p W B 9 8 0 through a fortuitous  A v r l l restriction site. T o ensure the tag  r e m a i n e d o n the protease, the A r g 2 4 3 S e r m u t a t i o n was i n t r o d u c e d into S G T to protect against potential autolytic cleavage. Y i e l d s o f S G T b e a r i n g a h e x a - h i s t i d i n e tag at the C - t e r m i n u s o f the p r o t e i n were three to five times l o w e r than the non-tagged construct. M a x i m a l y i e l d s o f r e c o m b i n a n t p r o t e i n b e a r i n g the tag d i d not e x c e e d 5 m g / L o f culture c o m p a r e d to 15 m g / L for the w i l d - t y p e construct. B i n d i n g o f the r e c o m b i n a n t protease to v a r i o u s c o m m e r c i a l l y a v a i l a b l e N i  + 2  or  +2 Co  -chelated resins was p o o r suggesting that the tag was p a r t i a l l y b u r i e d i n the p r o t e i n .  Inspection o f the c r y s t a l structure o f S G T suggests that the first t w o H i s residues m a y l i e i n a s h a l l o w cleft o n the e n z y m e surface. H o w e v e r , h i g h p u r i t y p r o t e i n resulted f r o m the p u r i f i c a t i o n ( F i g u r e 4.3). T h e r e d u c e d y i e l d o f S G T b e a r i n g the tag m a y h i n d e r f o l d i n g o f the e n z y m e . A l t e r n a t i v e l y , the tag m a y p r o m o t e interactions w i t h the n e g a t i v e l y c h a r g e d p e p t i d o g l y c a n f o u n d o n the c e l l w a l l o f the bacteria. H o w e v e r , the ease o f p r o t e i n p u r i f i c a t i o n 70  based o n the tag o u t w e i g h e d the d e m a n d for greater amounts o f protein. Y i e l d s o f each o f the mutants were s i m i l a r suggesting the mutations were not detrimental to p r o t e i n f o l d i n g . T h e s e results further support B.  subtilis as  an e x c e l l e n t e x p r e s s i o n host for the p r o d u c t i o n o f  proteases and other proteins that are d i f f i c u l t to produce i n  A  B  C  E. coli.  D  m - 2 3 kDa F i g u r e 4.3 P u r i f i c a t i o n o f a t y p i c a l H i s - t a g g e d mutant o f S G T f r o m B.  subtilis culture  u s i n g T a l o n m e t a l affinity resin. L a n e A , 1 m L supernatant; L a n e B , 1 m L c o l u m n f l o w through; L a n e C , 1 m L w a s h buffer. L a n e D : concentrated r e c o m b i n a n t p r o t e i n (2 Hg). S a m p l e s were concentrated u s i n g trichloroacetic a c i d i n lanes A - C .  4.3.2 T e c h n i q u e s f o r C h a r a c t e r i z a t i o n o f S u b s t r a t e S p e c i f i c i t y o f S e r i n e P r o t e a s e s C h a r a c t e r i z a t i o n o f the substrate s p e c i f i c i t y o f w i l d - t y p e F X a u s i n g c o m p r e h e n s i v e libraries o f s m a l l peptide substrates revealed the s e l e c t i v i t y o f the protease i s not strict [41,42]. Interestingly, the P 2 preference for large, planar h y d r o p h o b i c substrates over G l y side chains has been noted i n these studies. M o r e o v e r , P 3 and P 4 side chains are w e a k l y selected f o r . O n the basis o f this s p e c i f i c i t y , F X a is thought to resemble a l o w e f f i c i e n c y t r y p s i n rather than a h i g h l y selective t h r o m b i n [41]. O n e d r a w b a c k o f the libraries used to a n a l y z e the substrate s p e c i f i c i t y is the potential error caused by differences i n c o n f o r m a t i o n o f each o f the peptides i n w h i c h each peptide may adopt differing c o n f o r m a t i o n s and l e a d to bias.  71  In the present study, w e have used c o m m e r c i a l substrates that differ f r o m the natural p o l y p e p t i d e substrates o r short synthetic peptides c o m p o s e d o f L - a m i n o acids. T h e s e substrates s h o w e n h a n c e d s p e c i f i c i t y for m e m b e r s o f the c o a g u l a t i o n cascade a n d have b e e n w i d e l y used i n c l i n i c a l a p p l i c a t i o n s . N o n - s t a n d a r d a m i n o acids are present i n c l u d i n g o p t i m a l b l o c k i n g groups at their N - t e r m i n i to generate h i g h l y s p e c i f i c substrates ( F i g u r e 4.4). C o m p a r i s o n o f the rates o f h y d r o l y s i s o f these substrates s h o w s that the mutations created i n S G T have altered the active site g e o m e t r y and substrate s p e c i f i c i t y o f the e n z y m e .  4.3.3 Extended Substrate Specificity of SGT W i t h the T 1 9 0 P mutant o f S G T as the starting point, extended substrate s p e c i f i c i t y at the S 2 to S 4 p o s i t i o n s w a s i n t r o d u c e d b y substitution o f residues f o u n d i n F X a . T h e T 1 9 0 P mutant w a s c h o s e n o v e r the T 1 9 0 A m u t a t i o n for the h i g h e r K  m  values. A s the K  m  v a l u e for  m o s t peptides b e a r i n g the T 1 9 0 P mutant w a s a p p r o x i m a t e l y 5 0 to 100 p M , c h r o m o g e n i c substrates w o u l d be useful i n the k i n e t i c analysis o f mutants. In contrast, the T 1 9 0 A mutant o f S G T displayed K  m  values b e l o w 10 u M for each substrate. K i n e t i c d i s s e c t i o n o f subsequent  mutants based o n the T 1 9 0 A m u t a t i o n w o u l d require the use o f fluorescent substrates that are less c o m m o n l y u s e d for c o a g u l a t i o n proteases. M o r e o v e r , the T 1 9 0 P mutant p e r m i t t e d estimation o f the s p e c i f i c i t y constant ( k c / K ) u s i n g a d i r e c t interpretation o f the rate o f a t  m  h y d r o l y s i s o f peptide substrate. I n this m e t h o d , a s i n g l e substrate c o n c e n t r a t i o n that is w e l l b e l o w the K  m  v a l u e is h y d r o l y z e d a n d the rate measured b y spectrophotometry. T h e natural  l o g a r i t h m o f substrate r e m a i n i n g as f u n c t i o n o f t i m e i s p l o t t e d as a straight l i n e w h o s e s l o p e approximates k  c a t  / K . A n u m b e r o f mutants o f S G T w e r e c h a r a c t e r i z e d b y this m e t h o d a n d it m  w a s i n v a l u a b l e for d e t e r m i n i n g successful alterations o f substrate s p e c i f i c i t y .  72  S-2222  S-2238  Bz-lle-Glu(y-OR)-Gly-Arg-pNA R=H (50%) and R=CH3(50%)  H-D-Phe-Pip-Arg-pNA  S-2288  S-2302  H-D-lle-Pro-Arg-pNA  H-D-Pro-Phe-Arg-pNA  S-2366 pyroGlu-Pro-Arg-pNA  F i g u r e 4.4 Substrates used to characterize mutants o f S G T w i t h altered substrate s p e c i f i c i t y ( D i a p h a r m a , W e s t Chester, O h i o ) . N o n - s t a n d a r d a m i n o acids are incorporated into the substrate for i m p r o v e d d i s c r i m i n a t i o n a m o n g proteases.  73  4.3.4 Effect of the 99-loop on the Substrate Specificity of SGT Substrate s p e c i f i c i t y o f the S 2 b i n d i n g p o c k e t i n c o a g u l a t i o n proteases is affected b y the presence o f a t w o or three a m i n o a c i d i n s e r t i o n termed the 9 9 - l o o p . In F X a , the insertion o f t w o residues facilitates p o s i t i o n i n g o f T y r 9 9 and restricts access to the S 2 pocket. C h a r a c t e r i z a t i o n o f substrate s e l e c t i v i t y o f S G T revealed preferences inherent w i t h i n the protease ( F i g u r e 4.5). S p e c t r o z y m e P C a and S - 2 3 6 6 were preferred t w o - f o l d o v e r a l l other substrates u t i l i z e d . B o t h substrates have P r o at P 2 and this l i k e l y leads to presentation o f the A r g side c h a i n i n a c o n f o r m a t i o n m o r e favorable for h y d r o l y s i s . Importantly, the F X a preferred substrates were not preferred b y the T 1 9 0 P mutant o f S G T . T w o mutants were d e s i g n e d to s h o w the i m p o r t a n c e o f this l o o p i n generating s e l e c t i v i t y i n the S 2 p o c k e t ( F i g u r e 4.5). F i r s t , t w o residues, L y s 9 6 and G l u 9 7 , were i n t r o d u c e d to lengthen the 9 9 - l o o p i n S G T (denoted " L P b S G T " ) . A l l substrates e x a m i n e d were h y d r o l y z e d w i t h h i g h l y s i m i l a r r e a c t i o n rates to the T 1 9 0 P mutant u s i n g L P b S G T . A s this mutant presented the smaller, h y d r o p h i l i c side c h a i n T h r at p o s i t i o n 9 9 , no selectivity i n the S 2 b i n d i n g p o c k e t w a s anticipated. B o t h o f the inserted residues s h o u l d orient their c a r b o n y l o x y g e n s into the S 3 / S 4 p o c k e t i f the l o o p has the same c o n f o r m a t i o n o b s e r v e d i n other proteases. I n t r o d u c t i o n o f T 9 9 Y l e d to a n o n specific protease h a v i n g s i m i l a r preference for a l l substrates. T h e s e results suggested that the 9 9 - l o o p was i n a s i m i l a r c o n f o r m a t i o n to that observed i n F X a and that a d d i t i o n a l determinants o f substrate s p e c i f i c i t y were needed to reconstitute the desired s p e c i f i c i t y . N o t a b l y , substrates b e a r i n g P 2 G l y were m o r e e f f e c t i v e l y h y d r o l y z e d suggesting a s i m i l a r c o n f o r m a t i o n o f the l o o p to F X a . Structural s i m i l a r i t y o f the F X a 9 9 - l o o p is observed i n a P C . T h i s l i k e l y e x p l a i n s the m a i n t a i n e d preference for a P C preferred substrates ( S p e c t r o z y m e P C a and S-2366) i n each o f  74  the mutants c h a r a c t e r i z e d , i n c l u d i n g the mutant L P o f S G T . In a P C , residue 9 9 is also T h r . M u t a t i o n o f this p o s i t i o n i n a P C w i t h substitutions f o u n d i n F X a l e d to a protease w i t h a s i m i l a r a substrate s p e c i f i c i t y a n d i n h i b i t o r y profile to F X a . In the same study, the opposite mutations i n F X a ( Y 9 9 T ) l e d to a s i m i l a r s w i t c h i n g o f s p e c i f i c i t y [140]. I m p o r t a n t l y , as d e t e r m i n e d through peptide substrates, substrate specificity d i d not correlate w i t h the h y d r o l y s i s o f m a c r o m o l e c u l a r substrates and demonstrates the c r u c i a l r o l e for a d d i t i o n a l protein-protein interactions i n the c o a g u l a t i o n proteases.  M S-2266 • S-2366 • S-2222 •  SpectrozymeFXa  B SpectrozymePCa  • S-2302  T190P Figure 4.5 N o r m a l i z e d  kc /K a t  m  values for the T 1 9 0 P , L P and Y P mutants o f S G T .  V a l u e s d e r i v e d f r o m independent measurements d o n e i n t r i p l i c a t e ( ± 1 0 % S . D . ) . W e a k preference for a P C substrates is e x h i b i t e d by the e n z y m e i n i t i a l l y . I n t r o d u c t i o n o f the t w o residue l o o p a n d T 9 9 Y generates a b r o a d l y s p e c i f i c protease.  A n u m b e r o f other serine proteases have insertions at p o s i t i o n 9 9 . L a r g e insertions (greater than 5 residues) are f o u n d i n several k a l l i k r e i n s , the C l s protease, as w e l l as c o m p l e m e n t factor B [ 1 4 4 , 1 4 5 ] . C o m p a r e d to S G T , other proteases have a t w o o r three residue i n s e r t i o n at this r e g i o n i n c l u d i n g t h r o m b i n and pancreatic elastase II. In each o f these e n z y m e s , the l o o p p l a y s a r o l e i n d e t e r m i n i n g the substrate s p e c i f i c i t y o f the e n z y m e o f b o t h the S 2 and S 3 substrate b i n d i n g pockets. Increasing the length o f the i n s e r t i o n correlates w i t h  75  d e c r e a s i n g catalytic e f f i c i e n c y [145]. Futures studies for e n g i n e e r i n g substrate s p e c i f i c i t y s h o u l d a p p l y v a r i a b l e lengths o f a m i n o a c i d insertions and c o m p o s i t i o n s o f the 9 9 - l o o p .  4.3.5 Mechanisms of P3 Selectivity in S I Family Peptidases S e l e c t i v i t y for P 3 residues is p o o r i n nearly a l l S I peptidases. T h e enzyme-substrate interaction is l i m i t e d due to solvent e x p o s u r e o f the P 3 side c h a i n . I n F X a , P 3 b i n d i n g is generated b y the side chains o f residues 192 and 2 1 5 . T h r o u g h o u t the entire f a m i l y o f S I peptidases, residue 2 1 5 is h i g h l y c o n s e r v e d as a large planar side c h a i n ( W , F , Y ) . A l l c r y s t a l structures d e t e r m i n e d to date have s h o w n the side c h a i n i n a c o n f o r m a t i o n that borders the S 4 s p e c i f i c i t y p o c k e t ( F i g u r e 4.6). H o w e v e r , the b a c k b o n e c a r b o n y l g r o u p o f residue 215 is i n v o l v e d i n a h y d r o g e n b o n d w i t h the P 3 residue o f the substrate. In the c r y s t a l structures o f w i l d - t y p e r e c o m b i n a n t b S G T and T 1 9 0 P mutant, residue 192 d i s p l a y e d a h i g h degree o f f l e x i b i l i t y and was m o d e l e d as t w o c o n f o r m a t i o n s . F l e x i b i l i t y o f this residue has been demonstrated i n several c o a g u l a t i o n proteases a n d the alternate c o n f o r m a t i o n s facilitate interactions w i t h the P 3 and P 2 ' s p e c i f i c i t y p o c k e t s ( F i g u r e 4.7). A s b o t h S G T and F X a b o t h possess G i n residues at p o s i t i o n 192, m u t a t i o n was not r e q u i r e d . H o w e v e r , future w o r k s h o u l d i n v o l v e mutagenesis o f this residue due to its i n v o l v e m e n t i n substrate s p e c i f i c i t y and i n h i b i t i o n b y protease i n h i b i t o r s . R e s i d u e 192 has e v o l v e d to not disrupt substrate b i n d i n g and p l a y s a r o l e i n e n z y m e i n h i b i t i o n . Studies i n v o l v i n g p r o t e i n C , F X a , and t h r o m b i n have s h o w n that residue 192 is important i n protease-inhibitor contacts and is a k e y basis for differential i n h i b i t i o n [146-148]. M e t / G l u / G l n substitutions at p o s i t i o n 192 i n c o a g u l a t i o n factor F X a d i d not s i g n i f i c a n t l y alter substrate s p e c i f i c i t y u s i n g peptide substrates s i m i l a r to those used i n this study [143].  76  P r e s u m a b l y , m u t a t i o n o f the residue to a L y s side c h a i n i n S G T w o u l d generate an increased preference for a c i d i c side c h a i n s at P 3 . Unfortunately, L y s 192 has not been i n t r o d u c e d into any S I peptidases b y site-directed mutagenesis yet the residue exists n a t u r a l l y i n some m e m b e r s o f the f a m i l y .  170-1  Figure 4.6  S 3 & S 4 b i n d i n g pockets o f F X a . G i n 192 p l a y s a l i m i t e d r o l e i n  d e t e r m i n i n g the s e l e c t i v i t y o f the S 3 p o c k e t s t e m m i n g f r o m inherent f l e x i b i l i t y o f the side c h a i n . In the F X a structure w i t h o u t a substrate, the side c h a i n points a w a y f r o m S 3 pocket. T h e m o d e l depicted is the s u p e r i m p o s i t i o n o f F X a ( P D B I D 1 H C G ) and F V I I a i n c o m p l e x w i t h 1 , 5 - d a n s y l - G l u - G l y - A r g - c h l o r o m e t h y l ketone ( P D B I D 1 C V W ) , w i t h the F V I I a structure h i d d e n .  77  F i g u r e 4.7 S 3 a n d S 4 b i n d i n g p o c k e t s o f F V I I a . In the crystal structure o f h u m a n c o a g u l a t i o n factor V i l a i n c o m p l e x w i t h 1 , 5 - d a n s y l - G l u - G l y - A r g - c h l o r o m e t h y l ketone ( P D B I D 1 C V W ) , L y s 192 interacts w i t h the n e g a t i v e l y c h a r g e d side c h a i n i n P 3 o f the inhibitor.  S e q u e n c e analysis o f the S1 peptidases reveals a subset o f proteases that c o n t a i n p o s i t i v e l y c h a r g e d side chains at p o s i t i o n 192. T h e s e proteases s h o w S 3 s e l e c t i v i t y for a c i d i c P 3 residues. N o t a b l y , v e n o m b i n A and b i l i n e o b i n f r o m the m o c c a s i n snake  bilineatus) have L y s 192  (Agkistrodon  [149,150]. T h e s e proteases are f o u n d i n the v e n o m o f the snake and  78  m i m i c F X a b y a c t i v a t i n g t h r o m b i n at the same p o s i t i o n i n the p o l y p e p t i d e . A l t h o u g h c r y s t a l structures o f these proteases have not b e e n reported, m o l e c u l a r m o d e l i n g o f b i l i n e o b i n suggests that residue 192 o c c u p i e s a n i d e n t i c a l p o s i t i o n to that o b s e r v e d i n F X a , t h r o m b i n a n d S G T [151]. C r e a t i o n o f a n electrostatic interaction between the substrate a n d e n z y m e m a y decrease the rate o f d e a c y l a t i o n d u r i n g catalysis. Rates o f substrate h y d r o l y s i s b y L y s 192 b e a r i n g proteases are 10-fold l o w e r than o b s e r v e d f o r the c o a g u l a t i o n proteases. T h u s , the Q 1 9 2 K mutant o f S G T s h o u l d b e characterized a n d m a y y i e l d a stronger preference for a c i d i c side chains at P 3 w i t h a c o n c o m i t a n t r e d u c t i o n i n catalytic e f f i c i e n c y . H o w e v e r , it is l i k e l y that the h i g h f l e x i b i l i t y o f residue 192 w i l l l i m i t the m a x i m a l stringency f o r P 3 side chains. T w o m e c h a n i s m s have e v o l v e d to increase the selectivity o f the S 3 p o c k e t i n the S I f a m i l y o f peptidases. I n rat mast c e l l protease II, the absence o f a d i s u l p h i d e b o n d between residues 191 a n d 2 2 0 generates a d d i t i o n a l enzyme-substrate contacts a n d enlarges the S 3 p o c k e t [152]. P 3 selectivity c a n also b e generated b y a secondary p r o t e i n as evident i n the structure o f staphylokinase i n c o m p l e x w i t h the protease d o m a i n o f p l a s m i n . S t a p h y l o k i n a s e acts as a co-factor a n d inserts several side chains into the S 3 / S 4 pocket. A s a result the substrate s p e c i f i c i t y a n d i n h i b i t o r y p r o f i l e o f p l a s m i n are d r a s t i c a l l y altered [153]. T h u s , the serine protease scaffold is h i g h l y amenable f o r further e n g i n e e r i n g o f selectivity i n the S 3 b i n d i n g pocket.  4.3.6 Role of the 172-loop and Residue 217 in the SI Peptidases In order to f o r m the S 4 p o c k e t i n F X a , a stretch o f residues f r o m 172 to 174 adopt a c o n f o r m a t i o n distinct f r o m that o b s e r v e d i n b r o a d l y specific t r y p s i n l i k e e n z y m e s ( F i g u r e 4.8). I n S G T a n d other n o n - s p e c i f i c serine proteases, a large h y d r o p h o b i c side c h a i n at residue  79  172 buries itself into core o f the e n z y m e . In turn, the b a c k b o n e o f residues 173 and 174 adopts a c o n f o r m a t i o n that exposes the side chains o f these residues a w a y f r o m the S 4 pocket. In F X a , F I X a , t h r o m b i n a n d a P C residue 172 is T h r , M e t , o r Ser. I n these proteases the 1 7 2 - l o o p adopts an " u p " c o n f o r m a t i o n and bounds the S 4 pocket. In order to generate the proper c o n f o r m a t i o n o f the 1 7 2 - l o o p , t w o mutations were made i n S G T : Y 1 7 2 S and N 1 7 4 F .  F i g u r e 4.8 C o n f o r m a t i o n o f the 1 7 2 - l o o p i n S I peptidases: S G T ( A ) , F X a ( B ) , a P C ( C ) and F V I I a ( D ) . In F X a and a P C , the 172-loop adopts a c o n f o r m a t i o n s u c h that residue 172 is not b u r i e d i n the core o f the e n z y m e w h i c h is e v i d e n t i n a l l structures o f b r o a d s p e c i f i c i t y t r y p s i n - l i k e e n z y m e s . F V I I a has a large insertion at residue 170, a n d generates substrate s p e c i f i c i t y i n a different m a n n e r than other c o a g u l a t i o n proteases.  80  I n t r o d u c t i o n o f N 1 7 4 F alone into S G T y i e l d e d a protease w i t h s e l e c t i v i t y for S - 2 3 6 6 , a substrate d e s i g n e d for q u a n t i f i c a t i o n o f F X I a and a P C . B a s e d o n the c r y s t a l structure o f T 1 9 0 P S G T , N 1 7 4 F s h o u l d not affect the s p e c i f i c i t y o f the S 4 p o c k e t unless the c o n f o r m a t i o n o f the 1 7 2 - l o o p is distorted. T h e alteration o f substrate s p e c i f i c i t y m a y be due to the P h e side c h a i n at p o s i t i o n 174 r e p l a c i n g residue T y r l 7 2 its b u r i e d c o n f o r m a t i o n . Subsequent mutagenesis o f Y 1 7 2 S suggested that P h e 174 was i n a b u r i e d c o n f o r m a t i o n as it resulted i n a v e r y little change i n substrate s p e c i f i c i t y c o m p a r e d to the Y F P mutant o f S G T ( F i g u r e 4.9). A recent study reported the c r y s t a l structure o f rat a n i o n i c t r y p s i n w i t h s i m i l a r mutations i n the 1 7 2 - l o o p , and suggests the basis f o r a P C and F X I a - l i k e s p e c i f i c i t y [142]. In the c r y s t a l structure o f a mutant o f rat a n i o n i c t r y p s i n w i t h the S e r - S e r - P h e sequence substituted at the 172 to 174 p o s i t i o n s , the 172 l o o p adopts a n o v e l c o n f o r m a t i o n i n w h i c h P h e 174 buries i n w a r d i n the structure o f the e n z y m e s i m i l a r to that anticipated i n the mutants o f S G T [142]. A s a result, the c o n f o r m a t i o n o f the side chains at p o s i t i o n s 172 and 173 d o not m i m i c those o b s e r v e d i n F X a e v e n t h o u g h the p o l y p e p t i d e sequence is the same. T h e S 4 p o c k e t is enlarged as the 1 7 2 - l o o p extends farther a w a y f r o m the structure. I f a s i m i l a r c o n f o r m a t i o n o c c u r r e d i n the Y F P and Y S F P mutants o f S G T then the preference for substrates w i t h larger side chains at P 4 m i g h t be m o r e f a v o r e d . B o t h S - 2 3 6 6 and S - 2 2 6 6 have large h y d r o p h o b i c groups at P 4 ( p y r o g l u t a m i c a c i d and D - v a l i n e , r e s p e c t i v e l y ) .  81  1.00  090  • S-2266 • S-2366 • S-2222  080 0 70 0 60  050 0 40 0  so  0?0 0 10  000  YFP F i g u r e 4.9 N o r m a l i z e d kcJK  YSFP m  values for the Y F P and Y S F P mutants o f S G T .  Introduction o f Y 1 7 2 S y i e l d e d no substantial change i n substrate specificity. V a l u e s d e r i v e d f r o m independent measurements done i n triplicate ( ± 1 0 % S . D . ) .  A d d i t i o n o f the E 1 8 0 M m u t a t i o n to the Y S F P S G T mutant ( Y S F M P ) l e d to s i g n i f i c a n t i m p r o v e m e n t i n F X a - l i k e specificity ( T a b l e 4.3). In any k n o w n crystal structure o f an S I f a m i l y peptidase, the M e t side c h a i n at residue 180 makes no direct contacts w i t h the substrate or any c o m p o n e n t o f the protein. A l i m i t e d d i v e r s i t y o f a m i n o a c i d v a r i a t i o n is o b s e r v e d at p o s i t i o n 180 i n the S I peptidases w i t h a M e t residue present i n - 6 0 % o f the a l l proteins i n the f a m i l y . O n l y a few o f S I peptidases bear a p o s i t i v e l y c h a r g e d side c h a i n , s u c h as L y s or A r g , at this p o s i t i o n . P o s i t i o n i n g o f residue 180 is a c h i e v e d through a P-hairpin f o r m e d b y residues 177 to 180. T h e N H g r o u p o f residue 180 forms a h y d r o g e n b o n d w i t h the C = 0 b a c k b o n e o f residue 177. In S G T , the c a r b o x y l a t e m o e i t y o f G l u 180 also f o r m s a h y d r o g e n b o n d w i t h the N H g r o u p o f the amide b a c k b o n e o f V a i 177. M u t a t i o n o f residue 180 s h o u l d alter the electrostatic e n v i r o n m e n t o f the S 3 p o c k e t as w e l l as p e r m i t an alternate c o n f o r m a t i o n o f the 172-loop. D i s s e c t i o n o f the k i n e t i c constants o f the Y S F M P mutant b e a r i n g the E 1 8 0 M m u t a t i o n indicates moderate reconstitution o f F X a - l i k e properties. B o c - L e u - G l y - A r g was the most  82  specific substrate for the mutant protease i n d i c a t i n g the S 3 p o c k e t is h y d r o p h o b i c i n the mutant protease. Substrates d e s i g n e d for the q u a n t i f i c a t i o n o f F X a had the l o w e s t K  m  values  o f a l l substrates ( S - 2 2 2 2 and S p e c t r o z y m e F X a ) . H o w e v e r , substrates w i t h the l o w e s t K  m  values were not associated w i t h the highest turnover numbers. E f f i c i e n t turnover o f n o n - F X a preferred substrates still o c c u r r e d suggesting the extended b i n d i n g p o c k e t o f F X a was not present. B a s e d o n the observed properties o f the Y F P , Y S F P and Y S F M P mutants o f S G T , s t a b i l i z a t i o n o f the 1 7 2 - l o o p was i m p l i c a t e d as b e i n g necessary for generating F X a - l i k e extended substrate s p e c i f i c i t y . Further, c r y s t a l l o g r a p h i c analysis is r e q u i r e d to observe the c o n f o r m a t i o n o f the 1 7 2 - l o o p caused b y these mutations.  Substrate  K  m  (uM)  kcat  (min)  kc t/ K a  m  (uM" min") LGR B oc-Leu-Gly-Arg-pN A S-2222 Bz-Ue-Glu(Y-OR)-Gly-Arg-pNA (R=-HorCH at50%) S-2366 pyroGlu-Pro-Arg-pNAHCl  460 ±12  4664  FXa W K (uM min")  10.1  -  7.3  4.9  7.2  -  6.1  4.1  5.1  18.2  1.8  -  0.8  1.2  0.3  -  0.3  -  m  ±811  241  1765  ±36  ±959  511  3683  ±67  ± 1209  623  3788  ± 139  ±620  356  1800  3  Spectrozyme P C a H-D-Lys(g-Cbo)-Pro-Arg-pNA Spectrozyme F X a M-D-CHG-Gly-Arg-pNA S-2266 H-D-Val-Leu-Arg-pNA  ±20  ±240  2271  4017  ±468  ± 1687  S-2238 H-D-Phe-Pip-Arg-pNA  1396  1056  ±80  ±398  S-2302 H-D-Pro-Phe-Arg-pNA  1467  512  ±80  ±220  S-2251 H-D-Val-Leu-Lys-pNA  1857  527  ± 103  ±260  Table 4.3 Steady-state k i n e t i c parameters for the h y d r o l y s i s o f a series o f p - n i t r o a n i l i d e c h r o m o g e n i c substrates b y the Y S F M P mutant o f S G T c o m p a r e d to that o b s e r v e d w i t h F X a under s i m i l a r r e a c t i o n c o n d i t i o n s . ( F X a data f r o m ref. [140]). V a l u e s obtained i n triplicate ( ± S.D.).  83  W i t h i n the c o a g u l a t i o n proteases, residue 217 p l a y s a r o l e i n substrate selectivity. M u t a t i o n s at this p o s i t i o n have been characterized i n t h r o m b i n , c o a g u l a t i o n factor V i l a ( F V I I a ) , and c o a g u l a t i o n factor I X a ( F I X a ) [143,154,155]. In these studies, residue 217 was d e s c r i b e d as a determinant o f P 2 / P 3 s e l e c t i v i t y v i a f o r m a t i o n o f direct contact w i t h the substrate. C o n c l u s i o n s d r a w n were based o n analysis o f substrate s p e c i f i c i t y u s i n g o n l y a f e w peptide substrates and p r i m a r i l y through e n z y m e - i n h i b i t o r interactions e v i d e n t i n c r y s t a l structures. T h e latter c a n lead to misrepresentation o f the true f u n c t i o n o f p e r i p h e r a l residues o f the active site, such as residue 217, as the interaction between e n z y m e and i n h i b i t o r is t y p i c a l l y far tighter than for a natural substrate. M o r e o v e r , later studies established the s i g n i f i c a n c e o f a s o d i u m b i n d i n g site i n several o f the c o a g u l a t i o n factor proteases. B i n d i n g o f a s o d i u m i o n i m p r o v e s the catalytic e f f i c i e n c y o f the e n z y m e b y structural changes i n the protease d o m a i n . R e s i d u e 217 has been i m p l i c a t e d i n s t a b i l i z a t i o n o f the i o n b i n d i n g site [156]. Inspection o f c r y s t a l structures o f F X a , V i l a , and t h r o m b i n i n c o m p l e x w i t h peptide based i n h i b i t o r s supports neither o f these theories c o m p l e t e l y [ 1 4 1 , 1 5 7 , 1 5 8 ] . R e s i d u e 2 1 7 is t y p i c a l l y a n e g a t i v e l y charged s i d e - c h a i n throughout the S I f a m i l y o f peptidases, yet this residue is a T y r i n S G T . In several c r y s t a l structures o f c o a g u l a t i o n proteases, the c a r b o x y l g r o u p o f the G l u side c h a i n is i n v o l v e d i n the f o r m a t i o n o f t w o charge assisted h y d r o g e n b o n d s w i t h residue 173 i n the 172-loop. H e n c e , it w a s postulated that i n t r o d u c t i o n o f this side c h a i n w o u l d lead to s t a b i l i z a t i o n o f the l o o p i n an u p w a r d s c o n f o r m a t i o n and enhance F X a l i k e extended substrate s p e c i f i c i t y . K i n e t i c analysis o f the Y S F M P E mutant o f S G T b e a r i n g the G l u 2 1 7 y i e l d e d a protease m o r e s i m i l a r to F X I a and not F X a ( T a b l e 4.4). L i t t l e is k n o w n about the substrate s p e c i f i c i t y of F X I a  in vitro and  no crystal structure has been reported for the p r o t e i n . Inspection o f the  84  p o l y p e p t i d e sequence o f h u m a n F X I a s h o w s the presence o f M e t l 8 0 and G l u 2 1 7 , and a three residue insertion l o o p at p o s i t i o n 99 that w o u l d present either Ser or G l y into the S 2 p o c k e t [159]. C r y s t a l l o g r a p h i c analysis is r e q u i r e d to determine whether the i n t r o d u c e d G l u 2 1 7 side c h a i n adopts a c o n f o r m a t i o n that restricts the P 4 p o c k e t or whether it forms an electrostatic interaction w i t h A r g 2 2 2 . A s i n the Y S F P and Y S F M P mutations, the T y r l 7 2 i n the p o l y p e p t i d e sequence o f F X I a m a y possess a 1 7 2 - l o o p i n the d o w n c o n f o r m a t i o n . H e n c e , a l l o f the determinants thought to generate F X a - l i k e s p e c i f i c i t y are present i n F X I a w i t h the e x c e p t i o n o f T y r 9 9 and m a y e x p l a i n substrate s p e c i f i c i t y o f the Y S F M P E mutant o f S G T .  Substrate  K  m  (uM)  kcat (min)  kcat/ K m  (uM" min") S-2366 pyroGlu-Pro-Arg-pNAHCl  526  6691  ±23  ± 1409  S-2302 H-D-Pro-Phe-Arg-pNA  568  5487  ±39  ± 1569  Spectrozyme F X a M-D-CHG-Gly-Arg-pNA  167  819  ±37  ± 180  S-2266 H-D-Val-Leu-Arg-pNA  488  1624  ±33  ±343  Spectrozyme P C a H-D-Lys(g-Cbo)-Pro-Arg-pNA  1952  5870  ±34  ±1553  LGR Boc-Leu-Gly-Arg-pNA  177  196  ±22  ±86  S-2238 H-D-Phe-Pip-Arg-pNA  316 ±39  ±86  2341  1066  ± 126  ±701  S-2222 Bz-ne-Glu(y-OR)-Gly-Arg-pNA (R = H or C H at 50%)  198  FXa kcat/ K  12.7  (uM" min") -  9.7  -  4.9  18.2  3.3  -  3.0  4.1  1.1  -  0.6  1.2  0.5  4.9  m  3  Table 4.4 Steady-state k i n e t i c parameters for the h y d r o l y s i s o f a series o f p n i t r o a n i l i d e c h r o m o g e n i c substrates b y the Y S F M P E mutant o f S G T c o m p a r e d to that observed w i t h F X a under s i m i l a r r e a c t i o n c o n d i t i o n s . ( F X a data f r o m ref. [140]). V a l u e s obtained i n triplicate ± S . D .  85  4.3.7 Additional Elements Needed for Reconstructing FXa-like Specificity A d d i t i o n a l mutations must be made to i m p r o v e further F X a - l i k e s e l e c t i v i t y o f the S G T mutants constructed i n the present study. N o n - a d d i t i v e effects o f mutations are c o m m o n for mutations that are residues i n c l o s e p r o x i m i t y [153,160,161]. H e n c e , the absence o f one s p e c i f i c i t y determinant m a y h i n d e r the a b i l i t y o f other residues to f u n c t i o n p r o p e r l y . Studies attempting reconstitution o f F X a s p e c i f i c i t y i n other proteases m a y y i e l d clues as to what element is absent f r o m the Y S F M P E mutant o f S G T . U n f o r t u n a t e l y , these studies for the most part have f o c u s e d e x c l u s i v e l y o n the 9 9 - l o o p [70]. R a t a n i o n i c t r y p s i n ( R A T ) was mutated i n several o f the regions characterized i n this study but d i d not l e a d to an e n z y m e w i t h F X a - l i k e properties [142]. M u t a t i o n s o f residue 190, the 9 9 - l o o p , and the 1 7 2 - l o o p were c o m b i n e d i n R A T but were characterized o n the basis o f e n z y m e i n h i b i t i o n rather than substrate s p e c i f i c i t y . I n h i b i t i o n constants ( K ; ) o f i n h i b i t o r s o f F X a were t y p i c a l l y 10-fold h i g h e r w i t h the c o m b i n e d mutant o f R A T . O n the basis o f structure data, certain i n h i b i t o r s c o u l d assist the s t a b i l i z a t i o n the 1 7 2 - l o o p i n the proper u p w a r d s c o n f o r m a t i o n . B a s e d o n the data f r o m S G T mutants i n the present study, it appears that the c o n f o r m a t i o n o f the 1 7 2 - l o o p is the k e y i n g r e d i e n t m i s s i n g for reconstitution o f the e x t e n d e d substrate s p e c i f i c i t y o f F X a . S e v e r a l p o s s i b i l i t i e s exist for further s t a b i l i z a t i o n o f the 1 7 2 - l o o p . I n t r o d u c t i o n o f Y 2 1 7 E was unsuccessful at i m p r o v i n g the s e l e c t i v i t y o f the Y S F M P mutant o f S G T and m a y be due to the requirement o f a h y d r o x y l g r o u p at the side c h a i n o f residue 173, w h i c h is a G l y i n S G T . R e s i d u e s w i t h i n a 6 A radius o f residues 172 to 174 i n F X a i n c l u d e residues 167 to 176, 182, 215 to 2 1 7 , 2 2 4 and 2 2 7 . C o m p a r i s o n o f the c r y s t a l structures o f S G T and F X a s h o w that m o s t o f these residues are i d e n t i c a l and exist i n h i g h l y s i m i l a r c o n f o r m a t i o n s i n  86  b o t h proteases. Substitutions o f the residues adjacent to the 1 7 2 - l o o p i n the p o l y p e p t i d e sequence m a y further s t a b i l i z e the l o o p . H o w e v e r , the largest differences i n structure are evident at residues 2 2 4 and 2 2 7 . Importantly, these residues are i n v o l v e d i n the f o r m a t i o n o f the N a b i n d i n g site i n F X a . It is p o s s i b l e that i n t r o d u c t i o n o f a N a b i n d i n g site into +  +  Y S F M P E S G T w i l l reconstruct the e n z y m a t i c properties o f F X a . I f the site were present, b u r i a l o f residue 172 or 174 into the core o f the e n z y m e c o u l d be d i s f a v o r e d . Importantly, the Y S F M P E mutant has k i n e t i c properties s i m i l a r to F X I a w h i c h also does not possess a N a  +  b i n d i n g site. Sequence analysis o f the c o a g u l a t i o n proteases indicates that i n their e v o l u t i o n a r y past, a l l other c o a g u l a t i o n proteases h a d a N a b i n d i n g site. +  Site s p e c i f i c s o d i u m b i n d i n g has been demonstrated to p l a y a k e y r o l e i n the catalytic e f f i c i e n c y o f the c o a g u l a t i o n factor proteases i n c l u d i n g F X a , a P C , and t h r o m b i n . D i C e r a demonstrated the c r u c i a l r o l e o f residue 225 i n the serine proteases for b i n d i n g a single s o d i u m i o n near the active site o f the e n z y m e [162]. B i n d i n g o f s o d i u m and no other a l k a l i metal to these e n z y m e s generates a 3- to 5 - f o l d increase i n catalytic e f f i c i e n c y . In n e a r l y a l l serine proteases, residue 225 is o c c u p i e d b y a P r o and these proteases d o not demonstrate increased catalytic a c t i v i t y i n the presence o f s o d i u m i o n s . H o w e v e r , i n the c o a g u l a t i o n factor proteases, this residue is T y r . A b s e n c e o f P r o at p o s i t i o n 225 a l l o w s p r o p e r p o s i t i o n i n g o f the c a r b o n y l g r o u p o f the p r e c e d i n g residue i n the p o l y p e p t i d e sequence to b i n d the N a i o n [77]. +  In a d d i t i o n to residue 2 2 5 , an extensive n e t w o r k o f h y d r o g e n bonds and water m o l e c u l e s facilitates s t a b i l i z a t i o n o f a s o d i u m i o n i n a p o s i t i o n that is v e r y near the S 1 b i n d i n g p o c k e t ( F i g u r e 4.10). M u c h research has been devoted to the characterization o f the i o n b i n d i n g site i n t h r o m b i n , activated protein C and c o a g u l a t i o n factor X a [156,163,164]. D i s r u p t i o n o f any c o m p o n e n t o f the i o n b i n d i n g site r e a d i l y abolishes b i n d i n g and catalytic a c t i v i t y . T h e  87  c o m p l e x i t y o f these e n z y m e s has l i m i t e d o u r a b i l i t y to understand the structural a n d t h e r m o d y n a m i c effects o f s o d i u m b i n d i n g . H o w e v e r , the c r y s t a l structure o f t h r o m b i n i n the absence o f N a suggests that r e m o v a l o f the i o n destabilizes the entire S I p o c k e t and changes +  the c o n f o r m a t i o n o f the C y s l 6 8 - C y s l 8 2 d i s u l f i d e b o n d [165]. T h e S I p o c k e t lies adjacent to residue 172 i n S G T or residue 174 i n Y 1 7 2 S N 1 7 4 F mutants o f S G T i n the b u r i e d c o n f o r m a t i o n . Therefore, the c o n f o r m a t i o n o f the l o o p c o u l d be altered b y the presence o f an o c c u p i e d N a - b i n d i n g site +  F i g u r e 4.10  N a - b i n d i n g site i n t h r o m b i n . O c t a h e d r a l c o - o r d i n a t i o n stabilizes the i o n . +  B a c k b o n e c a r b o n y l groups and water m o l e c u l e s are i n v o l v e d i n the interaction. T h r e e ion-pairs s u r r o u n d the site and p r o v i d e stability.  Introduction o f a s o d i u m b i n d i n g site into a mutant S G T to i m p r o v e substrate specificity w o u l d i n v o l v e s i g n i f i c a n t mutagenesis o f the gene as nearly a l l o f the residues that  88  c o m p r i s e the site are absent i n S G T . In t h r o m b i n , the N a b i n d i n g site is l o c a t e d between t w o +  surface l o o p s b e g i n n i n g at residues 180 and 2 2 0 [77]. T h e i o n b i n d i n g site is 15-20 A distal f r o m the catalytic triad, yet lies w i t h i n 5 A f r o m D 1 8 9 . A c y l i n d r i c a l c a v i t y o c c u p i e d b y up to sixteen water m o l e c u l e s helps to stabilize the site. B o u n d N a is c o o r d i n a t e d octahedrally b y +  t w o c a r b o n y l o x y g e n atoms p r o v i d e d b y R 2 2 1 a a n d K 2 2 4 , and four b u r i e d water m o l e c u l e s . O n e o f these water m o l e c u l e s h y d r o g e n bonds to the side c h a i n o f D 1 8 9 e s t a b l i s h i n g a direct l i n k between the s o d i u m i o n and the S I site [52]. O v e r a l l stability o f t h e N a  +  site is p r o v i d e d  b y three i o n pairs, R 2 2 1 a - E 1 4 6 , K 2 2 4 - E 2 1 7 , and D 2 2 2 - R 1 8 7 . S o d i u m b i n d i n g i n v o l v e s residues adjacent to the substrate b i n d i n g p o c k e t and m a y p l a y a role i n generating stringent substrate s p e c i f i c i t y . In particular, residues 192 and 217 were b o t h targets for mutagenesis i n the present study and have been i m p l i c a t e d i n s o d i u m b i n d i n g .  4.3.8 Utility of a FXa-like Protease F a c t o r X a is r o u t i n e l y used for the cleavage o f r e c o m b i n a n t f u s i o n proteins, yet the preference for the I E G R cleavage sequence is not strict. F o r e x a m p l e , i n h o n e y bee p r e p r o m e l i t t i n , a related sequence, V L G R , was r e a d i l y c l e a v e d b y F X a [166]. A l t h o u g h this situation is not c o m m o n and c a n be prevented b y i n s p e c t i o n o f the p o l y p e p t i d e sequence o f the r e c o m b i n a n t protein, a m o r e specific protease is desirable. A n u m b e r o f vendors s u p p l y alternative proteases w i t h d i f f e r i n g r e c o g n i t i o n sequences, i n c l u d i n g T E V protease [167], t h r o m b i n [127], enterokinase [127], and P r e S c i s s i o n ™ protease [127] ( T a b l e 4.5). H o w e v e r , m a n y p l a s m i d s c u r r e n t l y e m p l o y e d c o n t a i n the F X a cleavage m o t i f in-frame w i t h c o m m o n restriction sites for c l o n i n g and r e c o m b i n a n t e x p r e s s i o n o f proteins. T o assess the a b i l i t y o f mutant S G T proteases to h y d r o l y z e the b o n d after the I l e - G l u - G l y - A r g sequence i n  89  m a c r o m o l e c u l a r substrates, p r o t h r o m b i n was digested w i t h l i m i t e d amounts o f each mutant constructed i n S G T ( F i g u r e 4.11). P r o t h r o m b i n was c h o s e n as it is the substrate o f F X a  in vivo  and has a n u m b e r o f potential cleavages sites.  Protease  Cleavage Sequence  Notes  Enterokinase  Asp-Asp-Asp-Asp-Lys  P I ' c a n not be a P r o .  Factor X a  Ile-Glu/Asp-Gly-Arg^  S e c o n d a r y cleavage sites (due to l o w P 3 and P 4 preference)  Thrombin  Leu-Val-Pro-Arg^Gly-Ser  S e c o n d a r y cleavage sites. C o n s i d e r a b l y m o r e e x p e n s i v e than F X a  T E V protease  Glu-Asn-Leu-Tyr-Phe-  L e s s useful for r e m o v a l  Gln^Gly  o f C - t e r m i n a l tags  PreScission  Leu-Glu-Val-Leu-Phe-  R e m a i n i n g sequence  protease  Gln Gly-Pro  after h y d r o l y s i s is  i  problematic  Table 4.5 C l e a v a g e sites o f proteases used i n p r o c e s s i n g r e c o m b i n a n t proteins.  A c c u r a t e p r o c e s s i n g o f p r o t h r o m b i n was not a c h i e v e d b y any o f the mutants constructed i n this study. O v e r n i g h t digestions are t y p i c a l l y used for site specific p r o t e o l y s i s o f r e c o m b i n a n t f u s i o n proteins and hence a s i m i l a r strategy was e m p l o y e d . C l o s e i n s p e c t i o n o f the d i g e s t i o n pattern s h o w n i n F i g u r e 4.11 shows that o n l y Y S F M P S G T y i e l d s a p r o t e o l y t i c fragment o f s i m i l a r size to the B - c h a i n o f t h r o m b i n . H o w e v e r , a d d i t i o n a l cleavages o c c u r and leave o n l y trace amounts o f the desired fragment. T h e Y S F M P E mutant o f S G T d i d not process p r o t h r o m b i n to y i e l d any fragment suggestive o f F X a - l i k e s p e c i f i c i t y and c o n f i r m s the data p r o v i d e d through h y d r o l y s i s o f s m a l l peptide substrates. P r o t h r o m b i n a c t i v a t i o n is a p o o r representative o f what w o u l d be anticipated for the cleavage o f t y p i c a l r e c o m b i n a n t proteins. In particular, several regions o f the p r o t e i n are o p t i m i z e d for h y d r o l y s i s  90  in vivo. Therefore,  it i s l i k e l y that the Y S F M P mutant o f S G T c o u l d be u s e d as a n alternative  for the site specific p r o t e o l y s i s o f r e c o m b i n a n t proteins as an alternative to F X a .  B —Prothrombin —Prethrombin 1 — T h r o m b i n B-chain — F r a g m e n t 1.2 —Protease —Fragment 1  j Fragment 1  Fragment 2  Fragment 1.2  F i g u r e 4.11  ~"1 Prothrombin  1  A-chain  B-chain  FXa Activation Fragments  P r o t h r o m b i n p r o c e s s i n g b y mutants o f S G T : T 1 9 0 P ( B ) , Y P ( C ) , Y S F P  ( D ) , Y S F M P ( E ) and Y S F M P E (F) mutants o f S G T c o m p a r e d to F X a ( G ) and undigested p r o t h r o m b i n ( A ) . O n l y the Y S F M P mutant o f S G T p r o c e s s e d trace amounts o f a p r o d u c t the same size as t h r o m b i n . T h e Y S F M P E mutant o f S G T contains a l l k n o w n specificity determinants o f F X a , but d i d not generate any o f the fragments associated w i t h the a c t i v a t i o n o f p r o t h r o m b i n .  91  4.4 Conclusions & Future Directions E x t e n d e d substrate s p e c i f i c i t y i n F X a is the result o f four a m i n o acids at p o s i t i o n s 9 9 , 1 7 4 , 1 8 0 , and 192. T h e s e residues are p o s i t i o n e d accurately b y residues 172 and 2 1 7 , and a t w o a m i n o a c i d i n s e r t i o n at p o s i t i o n 9 9 . O t h e r residues that surround these p o s i t i o n s are l i k e l y i n v o l v e d i n m i n o r o p t i m i z a t i o n o f the electrostatic e n v i r o n m e n t and stability o f the r e g i o n . S u b s t i t u t i o n o f the k e y residues o f F X a into S G T created a protease w i t h s i m i l a r substrate s p e c i f i c i t y that was m o r e s i m i l a r to F X I a rather than F X a . B a s e d o n these f i n d i n g s , the d e v e l o p m e n t o f a protease w i t h m o r e stringent s p e c i f i c i t y for the preferred F X a cleavage sequence m a y be p o s s i b l e t h r o u g h a d d i t i o n a l m u t a t i o n o f S G T p a r t i c u l a r l y b y a d d i t i o n o f the Na  +  b i n d i n g site as f o u n d i n F X a or t h r o m b i n . C e n t r a l to the success o f this research was the  use o f B. subtilis for p r o d u c t i o n o f the r e c o m b i n a n t p r o t e i n and mutants thereof. T h e ease o f p r o t e i n p u r i f i c a t i o n and l o w cost o f p r o d u c t i o n is a significant advantage o v e r p r e v i o u s l y reported systems. Structural and sequence s i m i l a r i t y o f S G T to F X a suggests that substrate s p e c i f i c i t y o f any c o a g u l a t i o n protease c o u l d be re-created i f not bettered. Increased stringency for the I l e - G l u - G l y - A r g F X a c o u l d be generated b y mutagenesis o f the 9 9 - l o o p and p o s i t i o n 192 i n the p o l y p e p t i d e sequence o f the Y S F M P E mutant o f b S G T . C h a r a c t e r i z a t i o n o f the s p e c i f i c i t y o f F X a s h o w s a w e a k preference at the P 2 , P 3 and P 4 p o s i t i o n s o f the substrate. O p t i m i z a t i o n o f the 9 9 - l o o p created i n S G T w i l l be needed to decrease f l e x i b i l i t y and restrict access to the S 2 pocket. H o w e v e r , it is u n k n o w n w h a t mutations w i l l create r i g i d i t y i n the 9 9 - l o o p . M u t a g e n e s i s o f residue 192 to a L y s a m i n o a c i d s h o u l d increase the s p e c i f i c i t y o f the S 3 p o c k e t for n e g a t i v e l y c h a r g e d side chains. Increased stringency i n the S 4 p o c k e t m a y be created b y strengthening the interaction o f residues 173 and 2 1 7 . In particular, mutants b e a r i n g Ser, T h r , or L y s at p o s i t i o n 192 m a y s t a b i l i z e the l o o p  92  further. E a c h o f these mutations w i l l . l i k e l y cause a decrease i n the catalytic e f f i c i e n c y o f the e n z y m e . T h e suggested a m i n o acids are l i k e l y not o b s e r v e d i n the w i l d - t y p e F X a p r o t e i n due to the p h y s i o l o g i c a l requirement o f e f f i c i e n c y . E v o l u t i o n has decreased the potential h a r m f u l effect o f less than perfect substrate s p e c i f i c i t y t h r o u g h l i n k a g e o f a d d i t i o n a l protein d o m a i n s that facilitate protein-protein and p r o t e i n - l i p i d interactions.  93  Chapter 5. General Discussion and Outlook  5.1 Substrate Specificity Determinants of the SI family of Serine Proteases Increasing the substrate specificity through mutagenesis o f the S I to S 4 p o c k e t s i n S G T was successful. S i x mutations were c o m b i n e d , a t w o residue i n s e r t i o n a n d five p o i n t mutations, to m i m i c the active site architecture o f c o a g u l a t i o n factor X a . O n l y the c o m b i n e d mutant demonstrated a strong preference for the desired I l e - G l u - G l y - A r g r e c o g n i t i o n sequence. Introduction o f a seventh m u t a t i o n i n S G T , Y 2 1 7 E , d i d not further i m p r o v e the s p e c i f i c i t y towards F X a preferred substrates. A s anticipated, s p e c i f i c i t y results l a r g e l y f r o m the a m i n o a c i d residues w h i c h constitute the enzyme-substrate interface but a d d i t i o n a l regions o f the protease are important. A further increase i n s p e c i f i c i t y w i l l require s t a b i l i z a t i o n o f the active site architecture a n d o p t i m i z a t i o n o f the electrostatic e n v i r o n m e n t o f the entire active site. T h e s e mutations w i l l i n v o l v e s e c o n d s h e l l o r m o r e distal residues t h r o u g h o u t the p r o t e i n . M o l e c u l a r e v o l u t i o n o v e r m i l l i o n s o f years has a c c o m p l i s h e d these tasks and has generated diverse proteases a n d substrate specificities. A n u m b e r o f proteases share s i m i l a r architecture a n d substrate s p e c i f i c i t y determinants as S G T . I n the M E R O P S database, S G T is c l a s s i f i e d as a m e m b e r o f C l a n S A , F a m i l y S I , S u b f a m i l y A peptidases [168]. N e a r l y 1000 proteases i n this f a m i l y h a v e b e e n identified, i n c l u d i n g the vertebrate a n d invertebrate c o a g u l a t i o n factor proteases, k a l l i k r e i n s , g r a n z y m e s , and, c o m p l e m e n t proteases ( F i g u r e 5.1). N o t a b l y , these proteases demonstrate t r y p s i n - l i k e , c h y m o t r y p s i n - l i k e , and elastase-like p r i m a r y substrate s p e c i f i c i t i e s ( T a b l e 5.1). A l t h o u g h the o v e r a l l architecture o f these proteases is s i m i l a r , v a r i a t i o n s i n the active site o f the e n z y m e facilitate differing specificities.  94  j— Insect Trypsins  i~L  SGT Invertebrate Trypsins  r Plasmin I L Vertebrate Trypsins , _ | ' — Vertebrate Chymotrypsins — Kallikreins I  I — Coagulation Factor Proteases I— C1r C1s Proteases Elastases  Granzymes & Cathepsin G r-S1A-  Insect Chymotrypsins Complement Factor B  — S1B—— Glutamyl endopeptidase \— S 1 C — P r o t e a s e Do S1-  S 1 D — L y s y l endopeptidase  — S 1 E — Streptogrisin A I— S1F —Astrovirus serine protease  F i g u r e 5.1 S i m p l i f i e d p h y l o g e n e t i c tree o f the S I f a m i l y o f peptidases. In each o f the S I sub-families o f peptidases, a diversity o f substrate specificities are f o u n d . S u b f a m i l y A is c o n s i d e r a b l y larger than the other sub-families w h i c h are l i m i t e d i n distribution to g r a m positive/negative bacteria and viruses.  95  Peptidases  Preferred PI Substrate  Granzyme A  R,K  Granzyme B  D  Granzyme H Granzyme M Human glandular kallikrein 2 Trypsin Plasmin Cruzain Duodenase  W,Y,F M,L R  S1' preference for S  [128]  R,K R,K Not P/I L,K,Y,F  Slight S1 preference for R > K Strong S1 preference for K > R Very broad specificity  [41,174] [41] [41] [175]  Trocarin  R>K  Notes  Ref.  Monomelic, dimeric and oligomeric structure [41,169] alters specificity P4:1 > V, P3: E > G; Preference for D at PI, and [41,170,171] results from residue 226 [172] [173]  Many snake venom proteases have specificities similar to coagulation factor proteases  [176,177]  Table 5.1 Substrate specificities o f S I f a m i l y peptidases.  C h y m o t r y p s i n - l i k e proteases require proper p o s i t i o n i n g o f large h y d r o p h o b i c side chains i n the p r i m a r y b i n d i n g p o c k e t for efficient catalysis. H e d s t r o m demonstrated that c o n v e r s i o n o f a t r y p s i n - l i k e e n z y m e to a c h y m o t r y p s i n - l i k e e n z y m e r e q u i r e d extensive mutagenesis o f the S I p o c k e t [56,57,59]. I n a d d i t i o n to p o i n t mutations at p o s i t i o n s 189, 2 1 6 , and 2 2 6 several l o o p s adjacent to the p o c k e t were r e q u i r e d to generate the change i n the p r i m a r y substrate s p e c i f i c i t y but resulted i n a p o o r l y active e n z y m e . Importantly, the altered l o o p s d o not contact the substrate d i r e c d y . I m p r o v e m e n t o f the catalytic e f f i c i e n c y against a m i d e substrates w a s a c h i e v e d b y mutagenesis o f Y 1 7 2 W [57]. I n the c o a g u l a t i o n proteases, the side c h a i n o f residue 172 i s e x p o s e d to the solvent a n d i n v o l v e d i n the S 4 substrate b i n d i n g p o c k e t [178]. I n the present study, m u t a t i o n o f residue 172 i n S G T w a s r e q u i r e d to m i m i c F X a - l i k e s p e c i f i c i t y o f the S 4 pocket. H o w e v e r , b u r i a l o f the aromatic side c h a i n at p o s i t i o n l e d to an alternate c o n f o r m a t i o n i n the 1 7 2 - l o o p . T h e s e studies demonstrate that m u l t i p l e a m i n o acids act i n concert a n d that s i m i l a r p o s i t i o n s i n the p o l y p e p t i d e sequence c a n affect d i f f e r i n g s p e c i f i c i t y p o c k e t s i n the f o l d e d protein.  96  A m i n o a c i d insertions i n the p r o t e i n sequence are a major c o m p o n e n t i n the substrate s p e c i f i c i t y o f serine proteases. H y d r o g e n b o n d s , d i p o l e m o m e n t s , steric constraints, and altered electrostatic environments c a n be generated b y the i n t r o d u c t i o n o f one or m o r e residues into the protein. In the present w o r k , a d d i t i o n o f a t w o residue l o o p at p o s i t i o n 99 w a s i m p o r t a n t i n altering the properties o f the S 2 pocket. L o o p insertions are f o u n d i n m a n y m e m b e r s o f the S I f a m i l y o f proteases. In a d d i t i o n to p o s i t i o n 9 9 , l o o p insertions are f o u n d at other p o s i t i o n s i n the protease sequence i n the S I f a m i l y peptidases. T h r o m b i n possesses a 10 a m i n o a c i d i n s e r t i o n at p o s i t i o n 6 0 as w e l l as a shorter l o o p at p o s i t i o n 148 that are i n v o l v e d i n substrate r e c o g n i t i o n [179,180]. T h e s e l o o p s have l i m i t e d f l e x i b i l i t y that has o n l y been demonstrated b y mutagenesis o f the protease [181]. O t h e r positions amenable for l o o p insertions i n c l u d e residue 170 f o u n d i n several c o a g u l a t i o n proteases [182], the " k a l l i k r e i n l o o p " at p o s i t i o n 9 0 [183], and also at residue 70 i n the neuropsins [184]. T h e l o o p s c a n also be i n v o l v e d i n b i o c h e m i c a l properties other than substrate s p e c i f i c i t y such as e n z y m e regulation, proteinprotein interactions and z y m o g e n a c t i v a t i o n . E x t e n d e d substrate s p e c i f i c i t y w i t h i n the S I f a m i l y o f peptidases c a n i n v o l v e residues o n the N - and C - t e r m i n i o f the scissile b o n d o f the substrate. Vertebrate c o a g u l a t i o n proteases possess substrate s p e c i f i c i t y i n the S I to S 4 pockets. S m a l l side c h a i n s at P I ' are also f a v o r e d b y m a n y proteases due to their decreased steric hindrance w i t h the e n z y m e . M e m b e r s o f the k a l l i k r e i n f a m i l y d i s p l a y s e l e c t i v i t y at P 2 ' for A r g side chains. R a t and h u m a n mast c e l l proteases have a m a r k e d preference for a c i d i c residues at P 2 ' due to the presence o f L y s 4 0 , A r g l 4 3 , and L y s l 9 2 [185]. H e n c e , it seems p o s s i b l e that a s i m i l a r strategy e m p l o y e d to  97  generate c o a g u l a t i o n factor X a s p e c i f i c i t y i n S G T c o u l d be a p p l i e d to generate proteases h i g h l y selective for a m i n o acids o n b o t h sides o f the scissile b o n d .  5.2 Molecular Evolution of the SI family of Serine Proteases O v e r the course o f e v o l u t i o n , proteases have been i n c o r p o r a t e d into a w i d e variety o f c e l l u l a r processes. A l t h o u g h the protease d o m a i n p r o v i d e s the catalytic m a c h i n e r y , a d d i t i o n a l protein d o m a i n s are l i n k e d to add functionality. Substrate r e c o g n i t i o n and c e l l u l a r (or extracellular) l o c a l i z a t i o n are t w o c o m m o n roles o f these d o m a i n s . F o r e x a m p l e , the C U B d o m a i n s facilitate protein-protein interactions [186], G i a d o m a i n s p r o m o t e p r o t e i n - l i p i d interactions [187] and fibronectin d o m a i n s l o c a l i z e proteins to e x t r a c e l l u l a r f i b r i n depositions [188,189]. B a s e d o n the d i v e r s i t y o f associated d o m a i n s , c o n s t r u c t i o n o f an accurate p h y l o g e n e t i c tree is a d i f f i c u l t task. A n u m b e r o f approaches have been taken to dissect the e v o l u t i o n o f the serine proteases. T h e s e studies c a n be d i v i d e d into those that e x a m i n e the entire protease sequence i n c l u d i n g the associated protein d o m a i n s , and those w h i c h e x a m i n e o n l y the protease d o m a i n or parts thereof [190-194]. A l l studies have supported the existence o f a s i n g l e ancestor for the entire S I f a m i l y o f peptidases [120]. A p p r o x i m a t e l y 4 0 a m i n o a c i d p o s i t i o n s are h i g h l y c o n s e r v e d throughout the f a m i l y to p r o d u c e a consistent three d i m e n s i o n a l structure. V a r i a t i o n s i n these residues are l i m i t e d to c o n s e r v a t i v e mutations that preserve the p o l a r i t y and size o f the side c h a i n [117]. T h e catalytic triad H i s - A s p - S e r is present i n a l l m e m b e r s o f the f a m i l y [195]. A c a r b o x y l - g r o u p c o n t a i n i n g a m i n o a c i d at p o s i t i o n 194 is also absolutely c o n s e r v e d amongst the f a m i l y . A s p / G l u l 9 4 p r o v i d e s the electrostatic interaction w i t h the N terminus o f the protease d o m a i n (residue 16) [196]. F o r m a t i o n o f this interaction stabilizes the  98  o x y a n i o n h o l e and the active site catalytic triad. L i m i t e d d i v e r s i t y at a p a r t i c u l a r residue and c l o s e p r o x i m i t y to the active site suggests i n v o l v e m e n t i n enzyme-substrate interactions and specificity. A n a l y s i s o f the a m i n o a c i d d i s t r i b u t i o n at each residue i n the substrate b i n d i n g p o c k e t s s h o w s a l i m i t e d n u m b e r o f p o s s i b i l i t i e s exist i n nature ( T a b l e 5.2). D e t e r m i n a n t s characterized i n the present w o r k (residues 180, 190, and 217) are m o d e r a t e l y c o n s e r v e d , and c a n be mutated further to y i e l d n o v e l proteases. O t h e r active site residues w i t h less sequence c o n s e r v a t i o n (residues 2 1 5 and 2 2 8 ) have not been characterized w i t h respect to substrate s p e c i f i c i t y but l i k e l y c a n be m a n i p u l a t e d to i n f l u e n c e specificity. T o g e t h e r w i t h the v a r i a b i l i t y i n size and c o m p o s i t i o n o f the l o o p s that surround the active site, the a b i l i t y to d e s i g n proteases w i t h specificities that d o not exist i n nature seems p o s s i b l e . A t present, it has not been established h o w h i g h l y specific proteases c o u l d be d e v e l o p e d i n the laboratory aside f r o m structure based d e s i g n . E l e m e n t s o f the present dissertation c o u l d be used i n s u c h a system.  5.3 Paper, Rock, Scissors Genetic Screening of Trypsin-like Proteases D e s i g n o f substrate s p e c i f i c i t y b y structure based techniques is l i m i t e d b y o u r i n a b i l i t y to understand the c o m p l e x interactions that o c c u r i n a p r o t e i n structure. M u t a t i o n s thought to e x h i b i t an effect often.result i n absent or o p p o s i n g results to that e x p e c t e d [125,197]. In the present study, b o t h T 1 9 0 P and Y 2 1 7 E mutations i n S G T y i e l d e d s i g n i f i c a n t l y different k i n e t i c properties f r o m w h a t were anticipated. T h u s , alternate approaches are r e q u i r e d . R a n d o m i z e d mutagenesis o f the w h o l e gene or parts thereof c o m b i n e d w i t h genetic s e l e c t i o n has been w i d e l y adopted for the a d d i t i o n or m a n i p u l a t i o n o f e n z y m a t i c properties.  99  Substrate Binding Pocket S2  S1 16  17  189  190  194  221  228  S3/P4  214  180  192  215  216  217  I  V  D  S  D  A  Y  S  M  Q  W  G  E  A.A.  72  60  69  36  99  50  72  93  61  46  63  90  14  %  V  I  S  A  E  G  F  T  Q  K  F  V  Y  A.A.  19  22  13  31  1  29  19  2  7  11  19  5  13  %  L  A  G  T  N  N  A  H  N  Y  A  V  A.A.  1  2  9  20  5  2  1  3  10  8  1  7  %  A  L  N  Q  S  E  F  H  I  L  A.A.  1  2  2  2  1  3  2  1  1  7  %  V  V  D  T  D  A  Y  S  E  Q  W  G  Y  SGT  I  V  D  A  D  A  Y  S  M  Q  W  G  E  FXa .  V  V  D  P  D  A  Y  s  M  Q  W  G  E  YSFMPE bSGT  T a b l e 5.2 Substrate s p e c i f i c i t y determinants o f S I f a m i l y s u b - f a m i l y A peptidases (not i n c l u d i n g l o o p regions). Sequence a l i g n m e n t o f 7 4 0 proteases i n the S I f a m i l y peptidases s h o w s a l i m i t e d d i v e r s i t y o f a m i n o acids exists at the substrate b i n d i n g r e g i o n i n a l l k n o w n m e m b e r s o f the f a m i l y (Sequence analysis d e s c r i b e d i n C h a p t e r 4.2.3). T h e final mutant construct ( Y S F M P E ) d i d not y i e l d the desired s p e c i f i c i t y , e v e n t h o u g h a l l k n o w n s p e c i f i c i t y determinants w e r e i n c o r p o r a t e d  C o n v e r t i n g t r y p s i n , or other p r i m i t i v e proteases, into h i g h l y s p e c i f i c e n z y m e s suffers f r o m the disadvantage that the w i l d - t y p e e n z y m e w i l l a l m o s t a l w a y s be m o r e active towards any substrate than the target e n z y m e . T y p i c a l directed e v o l u t i o n strategies e m p l o y creating n o v e l activities or substrate specificities that are c o m p l e t e l y disparate f r o m the w i l d - t y p e e n z y m e [198,199]. F o r e x a m p l e , the w i l d - t y p e e n z y m e does not catalyze a particular r e a c t i o n  100  or does so p o o r l y a n d the screen selects for i n c r e a s e d activity. S u c h a screen c a n not be a p p l i e d to increase p r o t e o l y t i c substrate s p e c i f i c i t y f r o m a p r i m i t i v e e n z y m e . A genetic screen for n o v e l substrate s p e c i f i c i t y m u s t also compensate for the large d i v e r s i t y o f p o s s i b l e substrates a r i s i n g f r o m 2 0 a m i n o acids at each p o s i t i o n . A three residue stretch o f a m i n o acids c a n have 8 0 0 0 different p o s s i b l e permutations. M o r e o v e r , the side c h a i n s have a degree o f s i m i l a r i t y that c a n not e a s i l y be accounted for. T h u s , i d e n t i f i c a t i o n o f the desired mutant p r o t e i n m u s t r e l y o n strategies e m p l o y e d i n nature. In particular, protease i n h i b i t o r s m a y be useful for i n f l u e n c i n g the m o l e c u l a r e v o l u t i o n o f proteases. R e c e n t m o l e c u l a r e v o l u t i o n o f H I V proteases serves as an e x c e l l e n t m o d e l for directed e v o l u t i o n to escape i n h i b i t i o n . F o r the v i r u s to r e p r o d u c e , a n u m b e r o f proteins are p r o d u c e d as precursors that m u s t be c l e a v e d for a c t i v a t i o n [ 2 0 0 , 2 0 1 ] . O n the basis o f this requirement, a n u m b e r o f protease i n h i b i t o r s have been d e s i g n e d and a p p l i e d c l i n i c a l l y i n the treatment o f this disease [202]. U n f o r t u n a t e l y , the v i r u s is k n o w n to mutate r a p i d l y a n d a n u m b e r o f mutations have been d e s c r i b e d that d i r e c t l y l e a d to resistance against i n h i b i t i o n [203]. T h e s e mutations are l o c a t e d at the active site cleft, as w e l l as at d i s t a l r e g i o n s o f the protease. N o t a b l y , resistance to i n h i b i t i o n p r o d u c e d altered substrate s p e c i f i c i t y o f the e n z y m e [204]. M u t a t i o n s i n the p r o t e i n substrates at the site o f c l e a v a g e have b e e n demonstrated to a c c o m p a n y the mutations that generate i n h i b i t o r resistance [205]. T h e s e observations suggest that e v o l u t i o n directed b y i n h i b i t i o n is a v a l i d c o n c e p t for the d e s i g n o f h i g h l y specific proteases. G e n e t i c screening for n o v e l substrate s p e c i f i c i t y c o u l d be generated b y c o m b i n i n g three c o m p o n e n t s : a protease, an i n h i b i t o r that affects the w i l d - t y p e but not the target protease, a n d a means b y w h i c h to v i s u a l i z e activity. Paper, R o c k , S c i s s o r s genetic screening  101  is put forth to a c c o m p l i s h these tasks ( F i g u r e 5.2). C o - e x p r e s s i o n o f an i n h i b i t o r w i t h the protease w i l l p r o v i d e the d i r e c t i o n for the e v o l u t i o n b y d i s t i n g u i s h i n g proteases that are s i m i l a r to the w i l d - t y p e i n the active site. V a r i a b l e s i z e d l o o p s at different p o s i t i o n s i n the p o l y p e p t i d e sequence o f S G T c o u l d be added and the w h o l e gene r a n d o m l y mutated b y various means [206,207]. O n l y active proteases w i t h altered active site geometry or properties w o u l d o v e r c o m e the i n h i b i t i o n and p o t e n t i a l l y y i e l d mutants w i t h i m p r o v e d substrate specificity. D e t e c t i o n o f protease a c t i v i t y c o u l d a p p l y a sensitive fluorescent substrate that is added d i r e c t l y to the s o l i d g r o w t h m e d i u m . F o r m a t i o n o f a h a l o w o u l d s h o w p r o t e o l y t i c a c t i v i t y and s i g n a l a p o t e n t i a l l y important mutant that c o u l d be characterized further. S i g n i f i c a n t t e c h n i c a l hurdles are e v i d e n t for this system to f u n c t i o n . In particular, the generation o f large mutant libraries i n  B. subtilis is s i g n i f i c a n t l y m o r e  and no k n o w n protease substrate w o u l d be amenable for  d i f f i c u l t than  E. coli  in vivo selection. T o o v e r c o m e  these  l i m i t a t i o n s n e w techniques and n o v e l proteins must be d e v e l o p e d .  E. coli trypsin i n h i b i t o r , e c o t i n , is a potent i n h i b i t o r o f t r y p s i n - l i k e serine  proteases  w i t h several properties amenable for use i n directed e v o l u t i o n . T h e b i n d i n g m o d e o f e c o t i n is i d e n t i c a l to that observed w i t h a natural substrate [208]. E c o t i n d i s p l a y s a n u m b e r o f useful properties, i n c l u d i n g thermostability and stability i n extreme p H . T h e i n h i b i t o r exists as a d i m e r i n s o l u t i o n and has t w o regions i n v o l v e d i n inhibitor-protease interactions ( F i g u r e 5.3) [209]. M a r k e d differences i n i n h i b i t o r y strengths are o b s e r v e d u s i n g the w i l d - t y p e p r o t e i n and h i g h s p e c i f i c i t y e n z y m e s ( T a b l e 5.3). C o a g u l a t i o n F a c t o r X a and t h r o m b i n have a 100- to 1 0 0 0 - f o l d l o w e r i n h i b i t i o n constant c o m p a r e d to b r o a d l y specific proteases [210]. A m o r e potent i n h i b i t o r against t r y p s i n - l i k e e n z y m e s c a n be constructed through mutagenesis o f the P I M e t to A r g [211]. In contrast, the i n h i b i t o r c a n be converted to a m o n o m e r i c f o r m w i t h  102  1000-fold less i n h i b i t o r y strength towards t r y p s i n - l i k e e n z y m e s [132]. T h u s , e c o t i n c a n be g e n e t i c a l l y m a n i p u l a t e d to e x h i b i t a b r o a d range o f i n h i b i t o r y strengths s p a n n i n g at least six orders o f magnitude. D i r e c t e d e v o l u t i o n to generate substrate s p e c i f i c i t y c o u l d use e c o t i n mutants iteratively, s u c h that the i n h i b i t o r y strength was i n c r e a s e d after e a c h r o u n d o f selection.  c  A c t i v e Protease Altered Specificity Inhibited Production of Halo  + +  + + +  +  -  +  -  F i g u r e 5.2 T h e o r y b e h i n d the Paper, R o c k s , S c i s s o r s genetic screen. A . I n i t i a l l y , the w i l d - t y p e protease is b l o c k e d f r o m h y d r o l y z i n g a substrate that is i n c l u d e d i n the s o l i d g r o w t h m e d i u m a n d no halo is evident. B . R a n d o m mutagenesis o f the protease gene w i l l lead to mutant proteases that escape i n h i b i t i o n and a h a l o s u r r o u n d i n g g r o w i n g bacterial c o l o n i e s w i l l signal potential clones. C . S e v e r a l p o s s i b i l i t i e s are accounted for b y this screen i n c l u d e the r e m o v a l o f non-active mutants and d i s c r i m i n a t i o n f r o m e n z y m e s w i t h w i l d - t y p e characteristics.  103  F i g u r e 5.3 D i m e r i c structure o f ecotin. T h e p r i m a r y b i n d i n g site interacts w i t h the target protease i n a c o n f o r m a t i o n i d e n t i c a l to that o b s e r v e d w i t h substrates. A secondary protease b i n d i n g site is p r o v i d e d b y the other c h a i n .  Protease  Kj (nM)  B o v i n e trypsin  <1  Factor X a  54  H u m a n l e u k o c y t e elastase  55  Human FXIIa  89  Human Kallikrein  163  T a b l e 5.3 I n h i b i t i o n constants o f ecotin against a variety o f S I f a m i l y peptidases. T h r o m b i n , activated protein C , tissue-type p l a s m i n o g e n activator and p l a s m i n are p o o r l y i n h i b i t e d b y w i l d - t y p e ecotin.  104  E c o t i n binds a protease i n a s i m i l a r f a s h i o n as a substrate and this property c o u l d be used to further c o n t r o l the directed e v o l u t i o n o f protease s p e c i f i c i t y . M u t a g e n e s i s o f the three residues p r e c e d i n g the P I residue c o u l d be used to alter the K i o f the i n h i b i t o r . T h e mutated sequence o f the protein w o u l d be the opposite o f the r e c o g n i t i o n site o f the protease. F o r e x a m p l e , m o n o m e r i c e c o t i n presenting G l n - L y s - T r p - M e t i n P 4 to P I s h o u l d p o o r l y i n h i b i t c o a g u l a t i o n factor X a w h i c h prefers the sequence U e - G l u - G l y - A r g . H o w e v e r , this mutant i n h i b i t o r s h o u l d still restrict the a c t i v i t y o f S G T . E c o t i n derives f r o m a bacterial source and p r o d u c t i o n o f the r e c o m b i n a n t i n h i b i t o r i n  B. subtilis s h o u l d be  high level o f recombinant protein expression i n  straightforward. G i v e n the  E. coli, y i e l d s i n B. subtilis s h o u l d p r o v i d e  sufficient m o l a r excess o f i n h i b i t o r . D e t e c t i o n o f a h i g h l y specific protease i n h i g h throughput screening requires a substrate that is w e l l defined, r e a d i l y quantified, and preferably i n e x p e n s i v e . A c o m m o n l y used m e t h o d for detection o f protease a c t i v i t y i n bacterial c o l o n i e s is the a d d i t i o n o f s k i m m i l k p o w d e r to the s o l i d m e d i u m . Protease a c t i v i t y leads to a z o n e o f clearance, o r h a l o , around the c o l o n y . U n f o r t u n a t e l y , h i g h s p e c i f i c i t y proteases d o not h y d r o l y z e s k i m m i l k effectively. A l t e r n a t i v e l y , peptide substrates s i m i l a r to that e m p l o y e d i n the present w o r k c a n be synthesized w i t h fluorescent l e a v i n g groups. H o w e v e r , these peptides are c o s t l y to p r o d u c e . G r e e n fluorescent protein ( G F P ) is c o m m o n l y used as reporter p r o t e i n for gene e x p r e s s i o n and c e l l u l a r l o c a l i z a t i o n and c o u l d be engineered for detection o f protease a c t i v i t y [212-214]. T h e fluorophore is generated f r o m three adjacent residues, S e r - T y r - G l y , that are sequestered o n the i n s i d e an 11-stranded p - b a r r e l ( F i g u r e 5.4) [215]. T h e p r o t e i n d i s p l a y s h i g h thermostability and extreme resistance to p r o t e o l y s i s r e s u l t i n g f r o m the t i g h t l y p a c k e d structure [216]. R e c e n t l y , W i l l i a m s d e s c r i b e d three regions i n G F P that c o u l d be rendered  105  a  sensitive to site specific p r o t e o l y s i s [217]. H o w e v e r , cleavage o f the p r o t e i n at any one o f these sites d i d not lead to a decrease i n  fluorescence.  These results w e r e l i k e l y due to the  stability o f the P - b a r r e l structure w h i c h d i d not u n f o l d after h y d r o l y s i s o f a s i n g l e b o n d . C o m b i n i n g two insertion mutations lead to a protein that d i d not f o l d p r o p e r l y , f o r m i n g i n c l u s i o n bodies i n their  E. coli expression  system, and further research w a s not c o n t i n u e d ( M .  W i l l i a m s , personal c o m m u n i c a t i o n ) . R a n d o m mutagenesis a n d s c r e e n i n g o f a d o u b l e insertion mutant o f G F P c o u l d be r e a d i l y performed to select for s o l u b l e p r o t e i n that  fluoresces  under  U V l i g h t [218]. G F P w o u l d then be a c o n v e r t e d to a useful reagent for the detection o f proteolytic a c t i v i t y surrounding a  in vivo.  A c t i v e protease w o u l d be e v i d e n t b y the f o r m a t i o n o f a halo  B. subtilis c o l o n y i f the  fluorescent  protein was added d i r e c t l y to the s o l i d  g r o w t h m e d i u m i n the p r o p o s e d screen.  F i g u r e 5.4 Structure o f G F P and potential regions for insertion o f a protease r e c o g n i t i o n sequence ( P D B code 1 E M A ) . F o r e x a m p l e , I l e - G l u - G l y - A r g - S e r inserted at positions 172 and 189 w o u l d a l l o w F X a to c l e a v e G F P t w i c e , r e l e a s i n g a strand o f the P - b a r r e l , and r e m o v i n g the  fluorescent  106  properties o f the m o l e c u l e .  In s u m m a r y , a directed e v o l u t i o n strategy for the d e v e l o p m e n t o f n o v e l p r o t e o l y t i c substrate specificities is p o s s i b l e . T w o proteins must be created and characterized p r i o r to v a l i d a t i n g s u c h an approach. T h e r e c o m b i n a n t e x p r e s s i o n o f S G T i n B . subtilis  provides a  useful starting p o i n t towards this g o a l and is a substantial i m p r o v e m e n t o v e r p r e v i o u s l y reported systems [59,98]. M u t a g e n e s i s o f the cleavage sequence presented b y G F P and the i n h i b i t o r w i l l p e r m i t screening for any s p e c i f i c i t y desired. N o v e l substrate b i n d i n g pockets m a y result that differ f r o m that o b s e r v e d elsewhere i n the f a m i l y , yet p r o d u c e s i m i l a r and m o r e stringent substrate specificities  5.5 F u t u r e O p p o r t u n i t i e s A n u m b e r o f n o v e l features exist i n the t r y p s i n scaffold that are p o o r l y understood and c o u l d be generated o n a s i m p l i f i e d s c a f f o l d such as S G T . T h e role o f s o d i u m b i n d i n g i n generating catalytic e f f i c i e n c y was p r e v i o u s l y d i s c u s s e d as a potential avenue for research. I n d u c e d fit m e c h a n i s m s o f substrate s p e c i f i c i t y have been d e s c r i b e d , p a r t i c u l a r l y i n the c o m p l e m e n t system [219]. A u t o l y t i c a c t i v a t i o n i n d u c e d b y receptor b i n d i n g is a w e l l k n o w n p h e n o m e n o n i n the vertebrate b l o o d c o a g u l a t i o n cascade [220]. Z y m o g e n a c t i v a t i o n m e c h a n i s m s are k n o w n to differ i n the S I f a m i l y peptidases [221-224]. L i n k a g e o f the protease d o m a i n to other p r o t e i n d o m a i n s c o u l d be studied to generate n o v e l f u n c t i o n and p r o t e o l y t i c activities. L a s t l y , little is understood i n the m e c h a n i s m o f catalytic rate enhancement caused b y p h o s p h o l i p i d and co-factor b i n d i n g i n the c o a g u l a t i o n cascade [225]. T h e s e m o l e c u l a r properties are l i k e l y not distinct m e c h a n i s m s acting alone. Structural p r o x i m i t y and direct interactions w i t h adjacent a m i n o a c i d side chains suggest c o m p l e x relationships w i t h substrate b i n d i n g and the catalytic process have yet to be f u l l y understood.  107  5.6 Significance of the Work In the present dissertation, a n o v e l e x p r e s s i o n system for t r y p s i n - l i k e e n z y m e s has been p r o d u c e d and o p t i m i z e d . P r e v i o u s studies have for the m o s t part used e u k a r y o t i c proteases w h i c h are inherently m o r e d i f f i c u l t to w o r k w i t h based o n their e v o l u t i o n to meet p h y s i o l o g i c a l f u n c t i o n . I have demonstrated that p r i m i t i v e t r y p s i n - l i k e e n z y m e s d e r i v e d f r o m a bacterial source c a n be used as a scaffold for e n g i n e e r i n g substrate s p e c i f i c i t y and f u n c t i o n a l properties s i m i l a r to e u k a r y o t i c proteases. A n u m b e r o f mutations were created i n S G T that increased the p r i m a r y s p e c i f i c i t y for A r g c o n t a i n i n g substrates and the extended substrate s p e c i f i c i t y was i m p r o v e d to p a r t i a l l y m i m i c F X a . T h e s e results s h o w that the substrate s p e c i f i c i t y o f any protease i n the S 1 f a m i l y o f peptidases c o u l d be d u p l i c a t e d u s i n g a s i m i l a r approach. Perfect m i m i c r y o f F X a substrate s p e c i f i c i t y was not a c h i e v e d l i k e l y due to the requirement o f a d d i t i o n a l s e c o n d s h e l l residues i n v o l v e d i n o p t i m i z a t i o n o f the b i n d i n g p o c k e t . Future w o r k c o u l d focus o n further r e p r o d u c t i o n o f F X a s p e c i f i c i t y t h r o u g h s i m i l a r a m i n o a c i d substitutions. A n alternative a p p r o a c h that i n v o l v e s r a n d o m mutagenesis and a n o v e l genetic screen has also been described that m a y y i e l d h i g h l y specific proteases that bear little sequence s i m i l a r i t y w i t h k n o w n proteases. D i r e c t e d e v o l u t i o n o f extended substrate s p e c i f i c i t y is t e c h n i c a l l y c h a l l e n g i n g and, i f successful, c a n s i g n i f i c a n t l y e x p a n d the repertoire o f protease t e c h n o l o g y . H i g h l y selective serine proteases w o u l d be useful i n a n u m b e r o f a p p l i c a t i o n s . T h e Y S F M P mutant o f S G T b e a r i n g six mutations c o u l d be used for the site s p e c i f i c p r o t e o l y s i s o f r e c o m b i n a n t proteins as an alternative to F X a . I n the future, it is anticipated that proteases c o u l d be d e s i g n e d w i t h levels o f substrate s p e c i f i c i t y a p p r o a c h i n g that f o u n d i n restriction  108  endonucleases [226]. T h e s e e n z y m e s c o u l d be used for h y d r o l y s i s o f proteins w i t h o u t the need for m a n i p u l a t i o n o f the D N A sequence for a d d i t i o n o f a protease r e c o g n i t i o n site. M o r e o v e r , peptide l i g a t i o n through reverse proteolysis has been described and c o u l d be c o m b i n e d w i t h h i g h l y selective mutants o f S G T [227]. T h u s , n o v e l proteins c o u l d be p r o d u c e d m o r e r a p i d l y i n an approach c o m p a r a b l e to c o m b i n a t o r i a l c h e m i s t r y .  5.7 C o n c l u s i o n s Substrate s p e c i f i c i t y o f the S I f a m i l y peptidases is d e r i v e d f r o m a f e w a m i n o acids i n the p r o t e i n sequence. D u e to the r e q u i r e m e n t o f s e c o n d shell residues for o p t i m i z a t i o n o f the substrate b i n d i n g site, structure based d e s i g n o f selectivity is a d i f f i c u l t yet a c h i e v a b l e g o a l . E n g i n e e r i n g s p e c i f i c i t y is then l i m i t e d b y w h a t exists i n nature. M o l e c u l a r e v o l u t i o n has not e x p l o r e d m a n y o f the p o s s i b i l i t i e s o f substrate s p e c i f i c i t y due to the p h y s i o l o g i c a l requirement for efficient catalysis. D i r e c t e d e v o l u t i o n , as p r o p o s e d i n the present dissertation or b y other means, is the next l i k e l y step i n the p r o g r e s s i o n o f protease t e c h n o l o g y .  109  Appendix A: Structural Alignment of Selected SI Family Peptidases Structures o f S I f a m i l y peptidases w e r e a l i g n e d u s i n g the c o m b i n a t o r i a l e x t e n s i o n m e t h o d a v a i l a b l e o n - l i n e at http://cl.sdsc.edu/ce.html [228]. C o n s e r v e d residues i n a l l 8 p o l y p e p t i d e s are denoted w i t h * a n d mutations constructed i n S G T are listed b e l o w the a l i g n m e n t . 60-loop 1SGT 1TLD 3 TGI 1PFX 1AUT 1HCG 1CVW 1PPB  1 1 1 1 1 1 1 1  WGGTRAAQGEFPFMVRLSM GCGGALYAQDIVLTAAHCV - --SGSGNNT--SITATGG IVGGYTCGANTVPYQVSLNSGYH FCGGSL INSQWWSAAHCY K S- -GIQVRLG IVGGYTCQENSVPYQVSLNSGYH FCGGSLINDQWWSAAHCY K S- -RIQVRLG IVGGENAKPGQFPWQVLLNGKIDA- - FCGGSIINEKWWTAAHCIEP- G -V- -KITWAG LIDGKMTRRGDS PWQVVLLDSKKKL - ACGAVLIHPSWVLTAAHCMDESK KLLVRLG IVGGQECKDGECPWQALLINEENEG- FCGGTILSEFYILTAAHCLYQAK RFKVRVG IVGGKVCPKGECPWQVLLLVNGAQ- - LCGGTLINTIWWSAAHCFDKI K NWRNLIAVLG IVEGSDAEIGMS PWQVMLFRKS PQELLCGASLISDRWVLTAAHCLLYP PWDKNF TENDLLVRIG 99-loop  I  I  1 SGT : 1TLD: 3TGI: 1PFX: 1AUT: 1HCG: 1CVW : 1PPB:  54 52 52 55 56 56 58 65  WDLQ- -S-G-AAVKVRSTKVLQAPGYN G-TGKDWALIKLAQPIN QPTLKIAT-T T EDNINWE-G-NEQFISASKSIVHPSYN-SNT-LNNDIMLIKLKSAASLNSRVASISLPT-S C EHNINVLE-G-NEQFVNAAKIIKHPNFD-RKT-LNNDIMLIKLSSPVKLNARVATVALPS-S C EYNTEETEP- -TEQRRNVIRAIPHHSYNATVNKYSHDIALLELDEPLTLNSYVTPICIAD-KEYTNI - F EYDLRRWE-K-WELDLDIKEVFVHPNYS-KST-TDNDIALLHLAQPATLSQTIVPICLPD-SGLAEREL DRNTEQEE-G-GEAVHEVEWIKHNRFT-KET-YDFDIAVLRLKTPITFRMNVAPACLPE-RDWAESTL EHDLSE-H-DGDEQSRRVAQVIIPSTYV- PGT -TNHDIALLRLHQPWLTDHWPLCLPERTFSERT-L KHSRTRYE-RNIEKISMLEKIYIHPRYN-WRENLDRDIALMKLKKPVAFSDYIHPVCLPD-RETAAS-L KE Y * * 96 99 -172-loop-  1SGT: 1TLD: 3TGI: 1PFX: 1AUT: 1HCG: 1CVW: 1PPB:  104 110 110 120 120 120 122 130  AYNQGTFTVAGWGA- NRE - GG SQQRYLLKANVPFVSDAACRSAY GNELVANEEICAGY ASAGTQCLISGWGN- TKSSGT SYPDVLKCLKAPILSDSSCKSAY PGQIT-SNMFCAGYAPAGTQCLISGWGN- TLSSGV NEPDLLQCLDAPLLPQADCEAS Y PGKIT - DNMVCVGF LK-FGSGYVSGWGR- VFNRG RSATILQYLKVPLVDRATCLRST KFTIY-SNMFCAGF NQAGQETLVTGWGY• HSSREKEAKRN-RTFVLNFIKIPWPHNECSEVM SNMVS - ENMLCAGI MT-QKTGIVSGFGR- THEKGRQS TRLKMLEVPYVDRNSCKLS S SFIIT - QNMFCAGY AF-VRFSLVSGWGQ- LLDRG ATALELMVLNVPRLMTQDCLQQSRKVGDS PNIT - E YMFCAGY LQAGYKGRVTGWGN- LKETWTANVGKGQPSVLQWNLPIVERPVCKDST RIRIT-DNMFCAGY* * S F M * ** 172 174 180  1SGT 1TLD 3TGI 1PFX 1AUT 1HCG 1CVW 1PPB  160 166 166 174 180 174 181 191  - PDT---GGVDTC- -QGDSGGPMFRK DNADEWIQVGIVSWGYGCARPGYPGVYTEVSTFASAI -L- E- --GGKDSC- -QGDSGGPWCS GKLQGIVSWGSGCAQKNKPGVYTKVCNYVSWI -L- E- --GGKDSC- -QGDSGGPWCN GELQGIVSWGYGCALPDNPGVYTKVCNYVDWI -H-E- --GGKDSC- -QGDSGGPHVTEVEGT-SFLTGIISWGEECAVKGKYGIYTKVSRYVNWI -L- G- --DRQDAC--EGDSGGPMVASFHGT-WFLVGLVSWGEGCGLLHNYGVYTKVSRYLDWI -D- T- - -KQEDAC- -QGDSGGPHVTRFKDT-YFVTGIVSWGEGCARKGKYGIYTKVTAFLKWI -S- D- --GSKDSC- -KGDSGGPHATHYRGT-WYLTGIVSWGQGCATVGHFGVYTRVSQYIEWL -K- PDEGKRGDAC- -EGDSGGPFVMKSPFNNR WYQMGIVSWGEGCDRDGKYGFYTHVFRLKKWI *p* ****E*** * ** * * ****** 190 217  1SGT 1TLD 3 TGI 1PFX 1AUT 1HCG 1CVW 1PPB  217 217 217 229 235 229 236 251  ASAARTL KQTIASN QDTIAAN KEKTK-HGHIRDK DRSMKTR QKLMRSE QKVIDQF  Streptomyces g r i s e u s t r y p s i n Bovine b e t a - t r y p s i n Rat a n i o n i c t r y p s i n Human coagulation f a c t o r IXa Human a c t i v a t e d p r o t e i n C Human coagulation f a c t o r Xa Human coagulation f a c t o r V i l a Human alpha-thrombin  110  % l d e n t i t y t o SGT 100 .0 35. 1 32 .7 34 .0 32. 2 32 .5 35. 9 33 .2  Ref. 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