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Mechanistic studies of xanthomonas manihotis b-galactosidase and the development of a rapid transglycosylation… Blanchard, Jan E. 2000

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M E C H A N I S T I C STUDIES OF XANTHOMONAS MANIHOTIS /3-GALACTOSIDASE A N D T H E D E V E L O P M E N T OF A R A P I D T R A N S G L Y C O S Y L A T I O N S C R E E N by Jan E . Blanchard B.Sc.; M c Master University, 1996 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E I N T H E F A C U L T Y O F G R A D U A T E S T U D I E S D E P A R T M E N T O F C H E M I S T R Y We accept this thesis as conforming to the^equiyed^sfandard March 2000 T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A © Jan E . Blanchard 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of fltemf^ft. The University of British Columbia Vancouver, Canada DE-6 (2/88) A b s t r a c t The Xanthomonas manihotis /3-galactosidase (BgaX) is a 66 kDa retaining gly-cosidase that hydrolyzes glycosidic bonds through a double displacement mechanism involving a covalent glycosyl-enzyme intermediate. Characterization of a recombinant form of this enzyme showed that it hydrolyzes pNPGal, DNPGal, pNPFuc, pNP-cu-L-Ara, pNPGalNAc, pNPGlc and pNPXyl with kinetic parameters in the range of kcat = 0.0217-36 s _ 1 and K m = 0.050-4.3 mM. The mechanism based inactiva-tor 2,4-dinitrophenyl 2-deoxy-2-fluorogalactopyranoside was shown to inhibit BgaX through the accumulation of a 2-deoxy-2-fluorogalactosyl-enzyme intermediate with the kinetic parameters kinact = 0.030 ± 0.004 s _ 1 and K; = 0.031 ± 0.005 mM. The fluorogalactosyl-enzyme intermediate was long lived, with a half life of 40 hr. Peptic digestion of this labeled enzyme and analysis by HPLC/mass spectrometry allowed the elucidation of G l u 2 6 0 as the catalytic nucleophile involved in the formation of the glycosyl-enzyme intermediate during catalysis. Retaining glycosidases are capable of catalysing the formation of glycosidic bonds through transglycosylation to an acceptor bound in the aglycone site of the enzyme. The second part of this study involved the development of a strategy to rapidly screen compounds for their potential as acceptors in transglycosylation reactions. This methodology was based on the premise that the reactivation, or turnover, of a gly-cosidase trapped as a 2-deoxy-2-fluoroglycosyl-enzyme is accelerated in the presence of a compound which productively binds to the aglycone site. The approach involved incubation of samples of the fluoroglycosyl-enzyme in the presence of a number of potential acceptors, followed by monitoring of the amount of enzyme reactivated due ii A B S T R A C T iii to transglycosylation at a fixed time. Using a 96-well plate format, seven glycosidases were screened in this manner using 46 different potential acceptors. Of the glycosides tested, 16-36% were positively identified as candidates to act as good acceptors for the given enzyme. Further evaluation of relative reactivation rates with these candidates was performed by monitoring the extent of reactivation of the fluoroglycosyl-enzyme species at a series of time points. The acceptors were ranked according to the observed initial velocity of reactivation. Generally, aryl glycosides and disaccharides were the preferred acceptors. Validation of the screening strategy as a method by which to identify good acceptors was carried out by the identification of products formed from some of the positively screened acceptors in four different cases. T a b l e o f C o n t e n t s Abstract ii Table of Contents iv List of Tables vii List of Figures viii List of Abbreviations x Acknowledgements xv Dedication xvi 1 Introduction 1 1.1 Glycosidases 1 1.2 Catalysis by glycosidases 3 1.2.1 The mechanisms of hydrolysis and transglycosylation 3 1.2.2 2-Deoxy-2-fluoro-glycosides: mechanism-based inactivators . . . 5 1.2.3 The identification of the catalytic nucleophile of a glycosidase 6 1.3 Glycosidases in oligosaccharide syntheses 7 1.3.1 Transglycosylation 7 1.3.2 Glycosynthases 10 1.4 The /3-galactosidase from Xanthomonas manihotis 13 1.5 Aims of study 16 iv T A B L E OF C O N T E N T S v 2 The Characterization of X. manihotis /3-Galactosidase 17 2.1 Introduction 17 2.1.1 Kinetic analysis of the hydrolysis reaction 18 2.1.2 Kinetic analyses of inactivation 18 2.2 Results and discussion 20 2.2.1 Purification 20 2.2.2 pH Stability . 22 2.2.3 pH Dependence 22 2.2.4 Hydrolysis kinetics 24 2.2.5 Glycone specificity 26 2.2.6 Inactivation by 2FDNPGal 30 2.2.7 The identification of the nucleophile of X. manihotis /3-galactosidase 34 2.2.8 The nucleophile mutant E260A BgaX 38 2.3 Conclusions 39 3 Screen of Potential Transglycosylation Acceptors 41 3.1 Introduction 41 3.1.1 Screening of potential acceptors 41 3.1.2 Preparative-scale transglycosylation reactions 43 3.2 Results and discussion 47 3.2.1 Large scale screening of potential transglycosylation acceptors . 47 3.2.2 The kinetics of enzyme reactivation via transglycosylation 53 3.2.3 Positively screened glycosides as transglycosylation acceptors 65 3.3 Conclusions 68 4 Materials and Methods 70 4.1 The characterization of BgaX 70 4.1.1 General 70 T A B L E OF C O N T E N T S vi 4.1.2 The molar absorptivity of 2,4-dinitrophenol and p-nitrophenol 71 4.1.3 Source, expression and purification of BgaX 72 4.1.4 pH Stability 74 4.1.5 pH Dependence 75 4.1.6 Kinetics 75 4.1.7 Identification of the catalytic nucleophile of BgaX 78 4.1.8 The mutant H6-E260A BgaX 79 4.1.9 Measurement of activity of H6-E260A BgaX . 79 4.2 Large-scale screening of potential acceptors 80 4.2.1 General 80 4.2.2 Screening A: large scale screening 81 4.2.3 Screening B: analylsis of enzyme reactivation 82 4.2.4 Preparative scale transglycosylation reactions 84 Bibliography 88 A Kinetic Analyses 93 A . l Basic enzyme kinetics 93 A.2 The Interpretation of kcat and kcat/Km 95 A.3 Inactivation 99 A.4 The Determination of Kj for a competitive inhibitor 100 A.5 Protection from inactivation 102 A. 6 Reactivation 103 B Graphical Representation of Data 105 B. l General •. 105 B.2 Enzyme kinetics 106 B.3 Enzyme reactivation 109 L i s t o f T a b l e s 2.1 Kinetic parameters of hydrolysis by BgaX 25 3.1 Positive hits from screened glycosides 50 3.2 Glycosides that are negative hits for all enzymes 51 3.3 Tranglycosylation reaction products from this study and the literature. 66 4.1 Values of e for pNP and DNP 72 4.2 Conditions for the measurement of activity of the enzymes screened. . . 80 4.3 Conditions for the screen of potential acceptors 82 4.4 Conditions for the analysis of the effect of each postive hit on enzyme reactivation 83 4.5 Conditions for preparative scale transglycosylation reactions 84 vii L i s t o f F i g u r e s 1.1 General oligosaccharide hydrolysis 2 1.2 The general mechanism of catalysis by a retaining /?-galactosidase. . . . 3 1.3 The general mechanism of catalysis by an inverting/3-galactosidase. . . 4 1.4 The oxocarbonium ion-like transition state 5 1.5 Fluoro-glycosides: mechanism-based inactivators 6 1.6 Transglycosylation 8 1.7 Azide rescue of the activity of a glycosynthase 11 1.8 Glycosidic bond formation via glycosynthases 12 1.9 Sequence similarities among family 35 glycosidases 14 1.10 Sequence similarities among family 35 and family 10 glycosidases. . . . 15 2.1 General scheme of enzyme catalysis . . . 18 2.2 General scheme of enzyme inactivation 19 2.3 Scheme showing protection from inactivation 19 2.4 The stability of BgaX with respect to pH 22 2.5 The dependence of kcat and kcat/Km of BgaX on pH 23 2.6 Structures of the pNP-glycoside substrates of Table 2.1 25 2.7 The inactivation of BgaX by 2FDNPGal 30 2.8 Ga/acto-configured pyridoimidazole (galacto-imidazole) 31 2.9 Protection of BgaX from inactivation 32 2.10 The spontaneous reactivation of 2-deoxy-2-fluorogalactosyl-BgaX. . . . 33 2.11 TIC of control and labelled peptides 35 2.12 MS/MS of the parent control and labelled peptides 37 2.13 Azide rescue of the activity of E260A BgaX 38 3.1 Reactivation of a glycosidase and subsequent re-inactivation by its transglycosylation product. 49 3.2 The reactivation of BgaX inactivated by 2FDNPGal 54 3.3 The reactivation profile of Man2A with pNP /?-xyloside 56 vm L I S T O F F I G U R E S ix 3.4 Summary of the reactivation profiles of BgaX 57 3.5 Summary of the reactivation profiles of BgaC 58 3.6 Summary of the reactivation profiles of Man2A 59 3.7 Summary of the reactivation profiles of Abg 60 3.8 Summary of the reactivation profiles of CelB 61 3.9 Summary of the reactivation profiles of Cex 62 3.10 Summary of the reactivation profiles of HBG 63 4.1 Primers for BgaX PCR 73 A . l The Michaelis-Menten curve 94 A.2 The Lineweaver-Burk (double reciprocal) plot 95 A.3 Reaction coordinate diagram for enzymatic catalysis 97 A.4 Lineweaver-Burk plots in the presence of a competitive inhibitor 101 A. 5 General plot of enzyme reactivation 104 B. l The determination of e of pNP and DNP 105 L i s t o f A b b r e v i a t i o n s G e n e r a l § section A 4 0 0 absorbance at 400 nm Ac acetyl BSA bovine serum albumin Da daltons DNP 2,4-dinitrophenyl e molar absorptivity (cm - 1 m M - 1 ) H6 6-histidine tag HPLC high-performance liquid chromatography LC/MS mass spectrometry in the single quadrupole scan mode Me methyl MS/MS mass spectrometry in the daughter ion scan mode m/z mass to charge ratio NMR nuclear magnetic resonance ODQOO optical density at 600 nm Ph phenyl j?NP para-nitrophenyl SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis LIST OF A B B R E V I A T I O N S xi SPh thiophenyl TIC total ion chromatogram TLC thin layer chromatography Kinetic Parameters kcat first order catalytic constant kcat /Km second order catalytic constant K m dissociation constant between enzyme and substrate •Ki dissociation constant between enzyme and inhibitor h.obs inact the observed rate of inactivation h-"'inact first order inactivation rate constant h.obs ^react the observed rate of reactivation T/react 0 the initial velocity of reactivation Glycosidases Abg Agrobacterium sp. /3-glucosidase Arthro Arthrobacter /3-galactosidase Bex Bacillus circulans xylanase BgaC Bacillus circulans /5-galactosidase BgaX Xanthomonas manihotis /3-galactosidase CelB Streptomyces lividans endoglucanase LIST OF A B B R E V I A T I O N S xii Cex Man2A H6-E260A BgaX HBG Strep C a r b o h y d r a t e s 2-deoxy-Glc 2-deoxy-Gal DNPCell DNPGal 2,5-DNPX 2 2FDNPCell 2FDNPGal 2FDNPGlc 2FGlcAF 2FManF a-Gal-F Gal-0-(l-3)-Glc-/3-SPh GaJ.-/3-(l-4)-Glc-0-SPh Cellulomonas ftmi xylanase/glucanase Cellulomonas fimi ^-mannosidase the 6-histidine tagged nucleophile mutant (Glu—>Ala) of BgaX human ^-glucuronidase Streptomyces coelicolor /3-galactosidase 2-deoxy-D-glucopyranose 2-deoxy-D-galactopyranose 2,4-dinitrophenyl /3-cellobioside 2.4- dinitrophenyl /3-D-galactopyranoside 2.5- dinitrophenyl /3-D-xylobioside 2,4-dinitrophenyl 2-deoxy-2-fluorocellobioside 2,4-dinitrophenyl 2-deoxy-2-fluorogalactopyranoside 2,4-dinitrophenyl 2-deoxy-2-fluoroglucopyranoside 2-deoxy-2-fluoro-/3-D-glucopyranuronyl fluoride 2-deoxy-2-fluoro-/3-D-mannopyranosyl fluoride a-D-galactosyl fluoride phenyl /3-D-galactopyranosyl- (1—>3) - 1-thio-/3-D-glucopyranoside phenyl /3-D-galactopyranosyl-(l->4)-l-thio-/3-D-glucopyranoside LIST OF A B B R E V I A T I O N S xiii galacto-imidazole a-Man-F M e - a - C e l l Me-/?-Cell MeXyl iV-Ac-glucalamine iV-Ac-glucosamine-/3-NH2 oNPXyl pAcPhGlc PhGlc pNP-a-L-Ara pNPCell pNPFuc pNPGal pNPGalNAc pNPGlc pNPGlcA pNPMan pNP-a-Xyl pNPXyl ga/acio-conflgured pyridoimidazole o>D-mannosyl fluoride methyl a-D-cellobioside methyl /3-D-cellobioside methyl /3-D-xylopyranoside 2-acetamido-l,5-anhydro-2-deoxy-D-ara6mo-hex-l-enitol 2-acetamido-2-deoxy-/3-D-glucosyl amine ori/io-nitrophenyl /3-D-xylopyranoside para-acetylphenyl /3-D-glucopyranoside phenyl /3-D-glucopyranoside para-nitrophenyl a-L-arabinopyranoside para-nitrophenyl /3-cellobioside para-nitrophenyl /5-D-fucopyranoside para-nitrophenyl /3-D-galactopyranoside para-nitrophenyl 2-acetamido-2-deoxygalacto-pyranoside para-nitrophenyl /3-D-glucopyranoside para-nitrophenyl /3-D-glucuronide para-nitrophenyl /3-D-mannopyranoside para-nitrophenyl a-D-xylopyranoside para-nitrophenyl /3-D-xylopyranoside LIST OF A B B R E V I A T I O N S xiv A m i n o A c i d s A Ala alanine M Met methionine C Cys cysteine N Asn asparginine D Asp aspartic acid P Pro proline E Glu glutamic acid Q Gin glutamine F Phe phenylalanine R Arg arginine G Gly glycine S Ser serine H His histidine T Thr threonine I He isoleucine V Val valine K Lys lysine W Trp tryptophan L Leu leucine Y Tyr tyrosine Acknowledgements First and foremost, great thanks goes to my supervisor Dr. Stephen Withers for his guidance and immeasurable patience. Thank you to Janine Foisey for cloning BgaX, and Dr. Laurent Gal for his help with protein expression as well as numerous insightful discussions. I greatly acknowledge Karen Rupitz for technical assistance, Shouming He for his expertise with mass spectrometry, Marietta Austria for generating my NMR spectra, and Helen Merkins and Drs. Tony Warren and Doug Kilburn for the use of their 96-well plate readers. I am very appreciative to all members of the Withers' group for the provision of various enzymes and inhibitors as well as many helpful discussions and suggestions. I thank them also for their friendship and support. Great thanks also go to my family who have encouraged me every step of the way, especially when that light at the end of the tunnel seemed so far away. Last, but far from least, I would like to thank Dan Melconian, my beau and best friend who has always been my inspiration. I never would have finished this without him. Thank you all. xv For my parents. Je t'aime. xvi C H A P T E R 1 I n t r o d u c t i o n 1 . 1 G l y c o s i d a s e s Carbohydrates, the most prevalent of all biological molecules [1], assume integral roles in vivo that are essential to a large number of cellular processes. Carbohy-drate monosaccharides are linked via glycosidic bonds to form a fantastic number of oligomers and polymers. These macromolecules serve a variety of functions both as polysaccharides (eg: structural enhancement, energy storage) and coupled with pro-teins and lipids in more complex conjugates (eg: recognition elements, extracellular matrix). Glycosyl hydrolases, or glycosidases, are enzymes that catalyse the hydrolysis of glycosidic bonds, and as such, play crucial roles in the catabolism of carbohydrates (Figure 1.1). Glycosidases are essential for processes which include post-translational modification of proteins in the rough endoplasmic reticulum, and digestion. The importance of glycosidases to proper cellular maintenance is evidenced by human dis-orders that are associated with the critical mutation of several of these enzymes, such as Tay-Sachs disease (deficiency in lysosomal hexosaminidase A), and human lyso-somal storage disease mucopolysaccharidosis type VII (mutation of /^-glucuronidase). 1 C H A P T E R 1 Introduction 2 O ' R O H + HOR v. V Glycone Aglycone F I G U R E 1 .1: G e n e r a l g lycos ida se c a t a l y s e d o l i g o s a c c h a r i d e h y d r o l y s i s . S u g a r h y d r o x y l s have b e e n o m i t t e d for c l a r i t y . As well as being an essential class of enzymes in vivo, glycosidases are also valuable di-agnostic tools in the elucidation of carbohydrate structure and function. Glycosidases are specific for the glycone glycosyl unit and its configuration at the anomeric center. The structure of an oligosaccharide can be determined by discovering which glycosi-dases are required to successively remove terminal glycosyl units from the oligomer. Once the overall structure of the oligosaccharide under study is determined, this same strategy can be applied to determine its function by the removal of glycosyl units and subsequent observation of any corresponding effects in vivo. Glycosidases have also proved useful in the synthesis of oligosaccharides through transglycosylation and the development of glycosynthases (vide infra). Large numbers of glycosidases have been isolated and characterized from a wide range of organisms thereby demanding the development of a well-ordered classification system. The primary distinction made within this class of enzymes is dictated by substrate specificity (core moiety and anomeric configuration) of the glycone portion of the active site [2]. More recently, a distinct classification of glycosidases has been developed that is based on amino acid sequence similarities [3, 4, 5]. Over two thousand glycosidases have been as-signed to over seventy families which share a common consensus pattern. Similarities in primary sequence demand that these proteins will fold and catalyse hydrolysis in a similar manner, thus making it possible to speculate as to the relatedness of en-C H A P T E R 1 Introduction 3 zymes within and between families. It also allows intelligent speculation regarding the identities of key catalytic residues, since candidates for these roles should be highly conserved. Interest in glycosidases is well founded and numerous studies, particularly over the past decade, have provided much insight into the characterization and mechanism of this class of enzymes. 1.2 Catalysis by glycosidases 1.2.1 The mechanisms of hydrolysis and transglycosylation The mechanism of glycosidase catalysis was first postulated by Koshland in 1953 [6] and has since been supported by structural, kinetic and mechanistic studies [7, 8, 9]. Substrates are hydrolyzed with either net retention or inversion of configuration at the anomeric center of the scissile bond with the aid of two strategically placed car-boxyl groups in the enzyme active site from either glutamic or aspartic acid residues. Retaining glycosidases function through a double displacement mechanism as shown R' HO H OR :0 Glycosylation Deglycosylation F IGURE 1.2: The general mechanism of catalysis by a retaining /3-galactosidase. C H A P T E R 1 Introduction 4 F I G U R E 1.3: T h e gene ra l m e c h a n i s m of c a t a l y s i s b y a n i n v e r t i n g /3 -galactosidase . in Figure 1.2. In the first step of catalysis (glycosylation) one of the carboxyl groups attacks the anomeric centre of the glycone sugar while the other provides general acid catalysis. This step results in the formation of a covalent glycosyl enzyme intermedi-ate that is then broken down by general base catalysed nucleophilic attack of either water (deglycosylation) or an 'acceptor' sugar (transglycosylation). Inverting glycosidases operate via a direct displacement mechanism with the act-ive-site carboxyl groups functioning as general acid/base catalysts (Figure 1.3). The transition states during catalysis by both retaining and inverting glycosidases have substantial oxocarbonium ion-like character (Figure 1.4). In the case of the retaining Agrobacterium sp. /3-glucosidase (Abg), secondary deuterium kinetic iso-tope effects have demonstrated that the transition state for deglycosylation is more oxocarbonium ion-like as compared to that of glycosylation [10]. C H A P T E R 1 Introduction 5 F I G U R E 1.4: T h e o x o c a r b o n i u m i o n - l i k e t r a n s i t i o n s ta tes for h y d r o l y s i s b y (a) r e t a i n i n g a n d (b) i n v e r t i n g g lycos idases . 1.2.2 2-Deoxy-2-fluoro-glycosides: mechanism-based inactiva-tors A class of mechanism based inactivators for retaining glycosidases has recently been developed in this laboratory [11, 12, 13]. These are glycosides that are substi-tuted with fluorine at the 2- or 5- position and have an activated leaving group, such as 2,4-dinitrophenyl (DNP) or fluoride, at the anomeric center. The 2- or 5-fluorine sub-stituent destabilizes the oxocarbonium ion-like transition states, slowing both steps of catalysis. However, the inclusion of a good leaving group at the aglycone site ac-celerates glycosylation. The net result of the attempted hydrolysis of such substrates by a retaining glycosidase is the accumulation of the glycosyl enzyme intermediate, rendering the enzyme inactive (Figure 1.5). It has been demonstrated through mass spectrometry, 1 9 F - N M R , and X-ray crystallography of glycosidases inactivated in this manner that the inhibited enzyme species is the covalent fluoro-glycosyl enzyme in-C H A P T E R 1 Introduction 6 O ^ O o . OHOH N 0 2 ) OHOH ° Y ° N02 D N P Y ° T H OH.OH °o^ Inactivation Reactivation F I G U R E 1.5: The inactivation of a /3-galactosidase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-/3-galactopyranoside (2FDNPGal) and subsequent reactivation, where kinact 3> kreact. termediate [12, 14, 15]. The existence of this intermediate is also suggested by the catalytic competence of the inhibited species. That is, once inactivated, a glycosidase will regain its catalytic capability over time as the result of the eventual turnover of the fluoro-glycoside. This reactivation can occur by the nucleophilic attack of water, as in the regular hydrolysis mechanism, or through attack by a suitable acceptor, as in transglycosylation. It is thus readily apparent that, since inverting glycosidases do not form a covalent glycosyl enzyme intermediate during hydrolysis, fluoro-glycosides are not effective inactivators of this class of enzymes. 1.2.3 The identification of the catalytic nucleophile of a glycosidase Fluoro-glycosides with good leaving groups have been shown to be excellent rea-gents with which to label the nucleophilic carboxylate of a retaining glycosidase, and C H A P T E R 1 Introduction 7 thereby allow the identification of the amino acid involved in nucleophilic catalysis. Once the enzyme is inactivated, the glycosyl enzyme species is partially digested by a protease and the resultant peptide fragments separated, and analyzed by mass spectrometry. The peptide covalently attached to the fluoro-glycoside is identified by comparison of the mass spectrum chromatogram to that of a control sample of unlabelled enzyme similarly treated, and sequenced to identify the catalytic amino acid residue. This procedure has been used successfully to assign the position of the catalytic nucleophile in many glycosidases [16, 17, 18, 19]. 1.3 Glycosidases in oligosaccharide syntheses The investigation of the functional roles of carbohydrates in vivo necessitates the synthesis of oligosaccharides for study. As simple as this statements sounds however, a synthetic scheme can quickly become quite cumbersome if the target in question is complex. Monosaccharides have several hydroxyls which may participate in glyco-sidic bonds, thereby requiring numerous protection and deprotection steps to attain the correct regio- and stereochemistry between units. Alternatively, an enzyme that catalysed the specific formation of glycosidic bonds could link glycosyl units in a single step reaction, thereby greatly facilitating oligosaccharide syntheses. 1.3.1 Transglycosylation Although retaining glycosidases preferentially hydrolyse their substrates in vitro, they have been exploited for their natural ability to transglycosylate. If transglycosy-lation can be enhanced relative to hydrolysis, glycosidases can catalyse the formation of glycosidic bonds in reasonable yields. For this reason, these enzymes have been C H A P T E R 1 Introduction 8 studied extensively to determine their utility in the synthesis of a number of target molecules (for an excellent review see [20]). The desired outcome of a transglycosylation reaction is the formation of a gly-cosidic linkage between a carbohydrate and another moiety, typically, although not necessarily, another sugar. The 'donor' in such a reaction is the source of the glycosyl unit to be transfered, while the 'acceptor' is the compound that becomes glycosylated. There are two basic approaches to inducing transglycosylation by glycosidases. In the 'thermodynamic' approach a large excess of donor and acceptor are used to favour the reverse of the hydrolysis mechanism to result in glycosidic bond formation. The yields associated with such reactions, however, are typically low (< 15%) owing to the inherent 55 M concentration of water that favours hydrolysis. Methods to favor transglycosylation involve the reduction of water content by the use of organic co-solvents, or the continuous removal of the synthesized product through adsorption onto activated charcoal or crystallization in situ. A second approach is that of syn-thesis under 'kinetic' control in which the glycosyl enzyme intermediate is intercepted •OR HO-OH.OH OH.OH OH NHAc OR FIGURE 1.6: Transglycosylation occurs when a suitable acceptor intercepts the glycosyl enzyme intermediate. For this illustrative example, donor = galactose (Gal) and acceptor = JV-acetyl-D-galactosamine (GalNAc.) C H A P T E R 1 Introduction 9 by the acceptor. Product yields using this strategy are often higher than those under thermodynamic control and this approach is therefore generally preferred. For the purposes of this report, 'transglycosylation' refers to those reactions that follow this latter approach. Potential donors for the kinetic approach are readily apparent from Figure 1.6 as they are necessarily those glycosides that will act as substrates for hydrolysis for the retaining enzyme in question. Donors are most often monosaccharides that are activated at the anomeric center, or simple disaccharides. As in hydrolysis, the newly formed linkage in the transglycosylation product has the same anomeric configuration as that of the donor sugar. The first criterion regard-ing which glycosidase should be selected as a catalyst for the synthesis of a specific linkage is thus readily apparent from the natural specificity of each glycosidase: for example, the coupling of a galactose residue to the non-reducing end of an oligosac-charide in a ^-linkage can be accomplished using an exo-/3-galactosidase. However, the further choice of an appropriate glycosidase based on aglycone specificity is not as simple. In order for product formation to occur, the acceptor moiety must bind produc-tively to the enzyme's aglycone site to stabilize the transition state of the transgly-cosylation step, thereby intercepting the glycosyl enzyme intermediate. Therefore, the greater the favorable interactions between the aglycone site and an acceptor, the greater will be the chance that transglycosylation will be favored over hydrolysis. This has been clearly demonstrated by the observation that the product yield of transgly-cosylation reactions varies widely with the identity of the acceptor employed [20, 21]. Further, the precise mode of interaction between the aglycone site and acceptor will determine which hydroxyl group attacks the glycosyl enzyme intermediate, thereby C H A P T E R 1 Introduction 10 defining the regiochemistry of the product. It is therefore evident that the speci-ficity of the aglycone site of a glycosidase is a strong determinant of the regiochemical outcome and yield of a transglycosylation reaction. The aglycone specificity of glycosidases has traditionally been examined using an approach in which the rates of hydrolysis of different substrates that share a common glycone moiety are compared. While this provides insight into the natural substrates of a glycosidase, it is not an adequate portrayal of acceptor specificity with respect to transglycosylation. Although substrate specificity with respect to hydrolysis may suggest several potential acceptors which could be used in transglycosylation reac-tions, it is not necessary that the best acceptors for a glycosidase be dictated by the susceptibility of their respective products. Further, the use of this strategy to examine aglycone specificity is not generally feasible as it requires access to a wide range of oligosaccharide structures due to the large number of unique acceptors used in various synthetic schemes. An alternative method to examine aglycone binding properties would be to carry out a series of transglycosylation reactions with various acceptors and determine which led to product formation via HPLC analysis. This process, however, would be very time consuming. A more reliable and speedy method by which to examine the specificity of the aglycone site of a glycosidase is therefore needed in order to ensure the choice of the most appropriate glycosidase for a desired transglycosylation reaction. 1.3.2 Glycosynthases Although transglycosylation by glycosidases has been shown to be useful in the synthesis of a number of targets, there are still several drawbacks to this methodology. The products of transglycosylation are necessarily substrates, and as such, may be C H A P T E R 1 Introduction 11 c r ^ 0 0 0 OHOH H N o 2 OHOH G - - " © C H 3 DNP C H 3 FIGURE 1.7: Azide rescue of the activity of a glycosynthase. rapidly hydrolyzed as they are formed, resulting in relatively low yields. The recent development of glycosynthases in this laboratory has afforded a new angle by which to maximize the utility of glycosidases for oligosaccharide synthesis. Glycosynthases are glycosidases that have been specifically mutated so as to replace the nucleophilic active site carboxylate with another amino acid residue, typically ala-nine or serine. X-ray crystallographic analyses have demonstrated that such mutant enzymes fold into the same wild-type tertiary structure and retain their active site ar-chitecture to successfully bind substrates as in the parent wild type protein [22]. Once bound however, the substrate cannot cleaved due to the alteration of the catalytic carboxyl. Activity with these mutants can be 'rescued' by supplying an appropriate nucle-ophile such as azide or formate (Figure 1.7) [23, 24, 25]. These small anions mimic the role of the missing carboxylate to aid in the cleavage of glycosides with activated aglycone moieties such as DNP. The utility of glycosynthases arises from their inability to catalyse hydrolysis cou-pled with their capacity to transglycosylate. If a glycosyl fluoride donor with the anomeric configuration opposite to that of the natural substrate is supplied, along C H A P T E R 1 Introduction 12 O' OH HO 'OH FIGURE 1.8: Glycosidic bond formation via glycosynthases. with a suitable acceptor, transglycosylation can occur as shown in Figure 1.8. The glycosyl fluoride donor mimics the glycosyl enzyme intermediate in the regular trans-glycosylation mechanism. Thus, if the appropriate donor and acceptor are supplied, a glycosynthase can catalyse transglycosylation, but not the hydrolysis of the product, ensuring a higher overall reaction yield. To date, 'successful' glycosynthases have been engineered by the replacement of the glutamic acid residue that functions as the catalytic nucleophile in three wild type glycosidases. Agrobacterium sp. /3-glucosidase (Abg) Glu358Ala [26] and Bacillus licheniformis l,3-l,4-/3-glucanase Glul34Ala [27] have been shown to catalyse trans-glycosylation in excellent yields (70%-90%), while Cellulomonas fimi /5-mannosidase (Man2A) Glu519Ala [28] appears to be much less efficient, with yields below 10%. Current studies with Abg are focused on the optimization of efficiency by the re-placement of the catalytic nucleophile with other amino acids. Results with the Abg Glu358Ser mutant show a 24-fold improvement in synthetic rates as compared to the alanine counterpart [29]. This increase in efficiency is presumably due to a stabilizing interaction between the hydroxyl of the side chain of serine and the departing fluorine of the donor sugar. This study also provides hope for the engineering of success-C H A P T E R 1 Introduction 13 ful glycosynthases from other glycosidases whose alanine mutants failed to catalyse transglycosylation. As seen with wild type glycosidases, the efficiency of product formation using glycosynthases varies with the chosen acceptor. Therefore, in order to maximize product formation, the most appropriate moiety with respect to aglycone specificity should act as the acceptor. Since glycosynthases retain the active site architecture of their parent wild type glycosidase, the aglycone specificity determined for the wild type enzyme should be applicable to its corresponding glycosynthase. Therefore, the evaluation of the specificity of the aglycone site of a wild type glycosidase would yield results that would also be applicable to syntheses using glycosynthases. 1.4 The /3-galactosidase from Xanthomonas mani-hotis The recently isolated /3-galactosidase from the pathogenic bacterium Xanthomonas manihotis (BgaX) is a 66 kDa exoglycosidase that hydrolyses (1—>-3) and, to a lesser de-gree (1—>4), linked terminal /3-galactose residues from oligosaccharides [30, 31]. Based on primary amino acid sequence similarities, BgaX has been classified as a family 35 glycosyl hydrolase (Figure 1.9) [31, 32]. This family consists of over 30 retaining /3-galactosidases, the vast majority of which are eukaryotic in origin. To date, there are only four bacterial members: BgaX, and /3-galactosidases from Bacillus circulans (BgaC) [32] Arthrobacter (Arthro) [33] and Streptomyces coelicolor (Strep) [34]. The significant sequence similarity between these prokaryotic enzymes and other /3-galactosidases of family 35 is of particular interest given the distant evolutionary relatedness of prokaryotic and eukaryotic species. Taron and coworkers have specu-C H A P T E R 1 Introduction 14 BgaX 153 A:Tli"ip'jA:L;A K Q fy.i Q ;P;L;L;N H;N;- - 1- I- A :V Q V QOSBEi - 8:T;A';DE|H A:Y;M;A D N 198 Bcir 126 A : Y > : D | V | L ] F E:R;LIR1PJIJIJS s[jj]-- B S M I J I J A ^ H I E E M ^ E - sgjb'JNHo gElO 170 42% 57%/553 Arthro 128 s Y-MIE> HQ A g i JLJV [P]R Q I D R - -.IJJJililL|vHiBHBBa - 1 :Y:G ;S | ]H H(Y]L]E Q(L] 1 7 1 35%! 51%/398 Strep 130 :.?M]g-P>IIilX^ - - EP3y;y>[vHiEBE03- s : Y ; G ; DUR A[Y]y|g]H;i; 174 37%, 51%/559 Human 158 | K | W | L | A | V | L | L | P | K | M | K | P | L | L | Y Qfiil- - iamv[TlT[vTilvlJ)l»CTas YI?1A-CB1P n^ YTrnRlp'rri 2 0 3 41%,52%/574 Mouse 159 IK|W|I.|O|V|LILIPIK[M1KTPTLTL1Y a j - - M«M Ji Ii iTfvHyBlllA'JJs Y I P I A ^ B I Y nlYlT.lRlpm 204 40%! 53%/574 Aspar 151 [K1F':T:E:K:I;V:S MIMIKIA E G L Y E T o leMJilili, S F 1 I U ) 1 A « 4 P V E Y Y F I G A A G ^ S Y 199 34%, 47%/318 Cat 159 |K|w|i.|GlvlLlLlp|k|M|KlP|L|L|Y Q[N1- - U J J T IT IT lv"n v FIJI s YIFIT C H Y DIYILIRIFILI 204 40%, 52%/576 BgaX Bcir Arthro Strep Human Mouse Aspar Cat |v GI3Y[W1AIGIW|F|D|HIWEI- K|P |H|A A T D A R Q Q 281 ;C M B F | W | H | G | W | F | D 1 H | W B - E E[H]H[T]R S A:E;S:y;254 L G[3_FjG L;A;G[F]E;P LE|Q D H(H]H[T]T S V Q E S 260 242 S | A | F | D K[L]I[K]F R[P]D Q Q R — . 215 S | A | F | A Q|L]K;°. Y Q[P"1H:AI3I.| jjiji216 S Q R N A|L|R[P CASH H Q T GAT H:V;ij i>|£jrji» ij ;A;W [rjt;;if *J|£JU u n\ttju. [Xj-i' s V u £ s 260 218 R[A]A A L | L | R | S R R[P]A E Q F F C A 13 F lw IN IG lw I F ID IH lw H - D K[H]H V R P A P S A 257 250 DIAIFIL s QlRlKlc E [P! K: G H L I 11 in is 13 F IY IT IG lw II ID IH lw B - Q|P |H|S[T1I K T:E;A:y';284 r^--- '-""'alalFlYlTlGlwlLlDlHlwB- KlPlHlsprlv K T K T:L;290 :"A275 250 DIAIFIL s QlRlKlc E |P1K jo Qi. I 251 QIAIFIL v Q | R | K | F iEJiloQH ii 236 C D;Y; F S P N K D N K Q K -M 249 A[A] Q I Q|R|K|S E[P1R;GI3L| iy" :T'I»1A[W1T|G|W|F|T G ! F Q G A V P Q R P A _ S'BFIYTTIGIWILIDIHIWH- QIPIHIS R V R T ;vly';290 F I G U R E 1.9: Sequence similarities among family 35 glycosidases using standard one letter amino acid abbreviations; codes for the origin of the enzymes are in the text or are self explanatory, except for Aspar, which is for asparagus. Identical amino acids are outlined with a solid line, similar with a dotted line, and conserved in white with a black background; dashes indicate gaps in the sequence. The top and bottom arrows indicate the position of the putative catalytic acid/base and nucleophilic amino acids respectively. Numbers at the beginning and end of the sequences indicate the amino acid numbering relative to the entire protein. Numbers to the right of the upper block of alignments indicate the total % identity and % similarity to BgaX, with the last value being the number of amino acids in the homologous region. C H A P T E R 1 Introduction 15 BgaX 72 [G]L N[T]V[E]T y v F | J 1 N L V | E | P | Q Q[G]Q[F]D[F|S G]N 97 BgaC 55 \G}F N |T]V[E]T Y V A 0 N L H | E | P | E E[G]Q[F]V[F]E S I 80 Strep 48 [G]L N A V;D;T Y V P|J1N F H[E]R T A[G]D I R[F]D H]p 73 Arthro 46 [G]L N[T]I [E]T Y v A | 3 N L H A[P]S E D V[F]D T S :A;G 71 Cal 65 D A I [T]P [E]N E M K | J I E V V | E | P | T E[G1N[F1D[F1T [G]T 89 Aka 67 |G]A L[T]P[E]N S M K [ 3 D A T | E | P | S R[G1Q[F]S[FIS [G]S 92 Tau 41 [G]Q V[T]P[E]N S M K E I D A T [ E | P | S Q[G1N[F~|N[F1A 66 Pch 71 G]Q L S P[E]N S M K E I D A T | E | P | S Q[G]Q[F]S[F]A G]S 96 Cfi 79 N L V V A[£ ]N A M K E I D A T | E | P | S Q N S[F]S[F]G A ;6 104 F I G U R E 1 10: Sequence similarities among family 35 and family 10 glycosidases. Identical amino acids are outlined with a solid line, similar with a dotted line, and conserved in white with a black background. Numbers at the beginning and end of the sequences indicate the amino acid numbering relative to the entire protein. Cal = Cryptococcus albidus xylanase A ; Aka = Aspergillus kawachii xylanase A ; Tau = Thermociscus aurantiacus xylanase; Pch = Penicillium chrysogenum xylanase; Cfi = Cellulomonas fimi cellulase/xylanase. lated that since X. manihotis is a plant pathogen, BgaX may have arisen as the result of gene transfer from a host [31]. However, the other three bacterial /3-galactosidases originate from non-pathogenic microorganisms and, as such, are less likely to undergo gene transfer with a eukaryotic species [33]. Another evolutionary possibility, also suggested by Taron and coworkers, is based on seven proposed regions of sequence homology of the enzymes of family 35. The first of these regions of the bacterial /3-galactosidases are similar to a domain found in family 10 glycosyl hydrolases (Fig-ure 1.10). These similarities therefore suggest that the prokaryotic enzymes may have evolved from a related xylanase. It is apparent that the inclusion of these bacterial en-zymes within a family of eukaryotic proteins poses interesting evolutionary questions as to how such similarities arose in such distantly related organisms. As noted in Figure 1.9, BgaX has a significant degree of sequence similarity with a human lysosomal /3-galactosidase. Mutations at several sites in this human enzyme C H A P T E R 1 Introduction 16 result in the neurological disorders GMl-gangliosidosis and Morquio B. syndrome [35]. Due to the degree of similarity between the two enzymes, mechanistic or structural studies of BgaX are of particular interest as they could contribute to an understanding of the human /3-galactosidase and its associated disorders. Hydrophobic cluster analysis, coupled with primary amino acid sequence similari-ties, has predicted the position of the nucleophilic and acid/base amino acid residues of the glycosidases of family 35 [36]. As shown in Figure 1.9, these analyses predict the catalytic nucleophile of BgaX as G l u 2 6 0 . The assignment of the postition of the corresponding carboxylate in the human /3-galactosidase has been experimentally con-firmed in this laboratory by the previously described labeling methodology utilizing a 2-deoxy-2-fluoro-glycoside (§1.2.3) [18]. However, given the diversity of this family, it seemed wise to confirm the identity of the nucleophile in an enzyme from a prokaryotic source. 1.5 A i m s of s t u d y The aims of this study were two-fold: (i) Identify the catalytic nucleophile of BgaX through a labeling study utilizing a 2-deoxy-2-fluoro-galactoside. (ii) Develop a simple, yet rapid, screening procedure that identifies the most suit-able acceptors for a given glycosidase or glycosynthase for transglycosylation reactions. C h a p t e r 2 The Characterization of X . m a n i h o t i s /3-Galactosidase 2 . 1 I n t r o d u c t i o n It was noted in §1.4 that the assignment of the catalytic nucleophiles of family 35 glycosidases has been experimentally demonstrated to be accurate in the case of the human lysosomal /3-galactosidase. Although there is a significant degree of global primary sequence homology among enzymes of this family, the local region bracketing the putative active site nucleophilic carboxylates appears to be somewhat variable (Figure 1.9). Greater sequence conservation can be seen within the region encompassing the residue that functions as the catalytic acid/base. Sequence similarity to the human /5-galactosidase was used to assign the putative sites of the catalytic residues of BgaX as G l u 2 6 0 (nucleophile) and G l u 1 8 0 (acid/base). Given the weak sequence conservation in the region of the catalytic nucleophile, it was of interest to experimentally verify the position of this active site residue. As well as identifying a catalytic residue of BgaX, we desired to examine the aglycone specificity of this enzyme with respect to transglycosylation reactions. These 17 C H A P T E R 2 The Characterization of X. manihotis /3-Galactosidase 18 results are reported in the following chapter which discusses the development of the aglycone screening method. We also briefly examined the specificity of the glycone site of BgaX through the kinetic analysis summarized below (see Appendix A for details). 2.1.1 Kinetic analysis of the hydrolysis reaction Enzyme catalysis can be described by the following scheme: k_\ kcat £ _ j + 1 E + S ^ ^ E-S E + P K m = F I G U R E 2.1: General scheme of enzyme catalysis, where E is the free enzyme, S the substrate, E«S the Michaelis-Menten complex, and P the final product. Values for kcat and K m are determined by measuring the initial reaction velocity at several substrate concentrations, and subsequent fitting of the data to the classical Michaelis-Menten expression: The parameters kcat/Km and kcat are rate constants that are related to the first irreversible step of catalysis and the rate determining step respectively. Comparison of these kinetic parameters for different substrates therefore provides insight into substrate specificity and the energy barriers associated with catalysis. 2.1.2 Kinetic analyses of inactivation The inactivation of an enzyme by a mechanism-based inactivator can be described by the following general scheme: C H A P T E R 2 The Characterization of X. manihotis (3-Galactosidase 19 E + I inact El k_i + k inact F I G U R E 2.2: General scheme of enzyme inactivation where E and I are the free enzyme and inhibitor, E»l the Michaelis-Menten complex, and El the inactivated enzyme species. The parameters kinact and Kt are calculated from the observed rates of inactivation (Knact) a t several different inhibitor concentrations through fitting of this data to a relationship analogous to the Michaelis-Menten expression (Equation 2.1): kinactl}] h.obs _ inact ^ + m (2.2) The illustration that an inactivator is active site-directed can be achieved by mon-itoring the rate of the inactivation of the enzyme in the presence of an additional competitive inhibitor. This scenario is summarized in Figure 2.3 where P is the com-petitive inhibitor. E + + P k'i k'_ E«P r E- l K/ + k El inact F I G U R E 2.3: Scheme showing the protection of an enzyme from inactivation by a mechanism-based inactivator in the presence of a competitive inhibitor. The validity of applying this model to the inhibition of a glycosidase by a 2-deoxy-2-fluoro-glycoside can be determined by the ratio of the observed rate of inactivation in the presence (&fpS) and absence (k°bs) of a suitable competitive inhibitor: C H A P T E R 2 The Characterization of X. manihotis (3-Galactosidase 20 uobs fcobs Kt + [/] * ( l + j g ) + [ 7 ] (2.3) 2.2 R e s u l t s a n d d i s c u s s i o n 2.2.1 Pur i f ica t ion A tag of six histidine residues was incorporated into the BgaX protein at the carboxy terminus through direct manipulation of the gene. This modification made it possible to employ a Ni2+-chelating column in the first step of the purification of overexpressed BgaX from E. coli thereby eliminating much extraneous cellular material in a single step. The remaining impurities were removed by pooling those fractions containing /?-galactosidase activity and subsequent application to a cation-exchange column. The resultant fractions that contained BgaX were combined into a single preparation that had a specific activity of 28.3 mol/min/mg with pNPGal at pH 6.0, and was homogeneous by silver stained SDS-PAGE. • In the first step of purification, BgaX was eluted from the Ni2+-chelating column by a linear gradient of 0-100 mM imidazole. Although imidazole was effective at eluting BgaX from the column, its presence appeared to induce the aggregation of a portion of the desired enzyme (as determined by silver stained SDS-PAGE of the observed precipitate). Even over the time course needed to remove the imidazole by dialysis, a precipitate was observed to form. Once dialysis was complete, no addi-tional precipitation was observed in the subsequent purification step or in the final concentrated enzyme preparation. Exactly why imidazole would cause this aggrega-tion is unclear, but it is possible that a high concentration of this compound could C H A P T E R 2 The Characterization of X. manihotis /3-Galactosidase 21 have raised the ionic strength of the preparation to such a degree as to salt the enzyme out of solution. Although some BgaX was lost through precipitation, the amount of purified en-zyme isolated from the second step of the protocol (typically 30 mg/2L culture of E. coli) was sufficient for our purposes. Therefore, although high concentrations of imi-dazole appear to result in the loss of some enzyme, the use of a Ni2+-chelating column was seen as an appropriate initial step by which to eliminate most of the extraneous proteins from the preparation. It is apparent however that if this procedure is to be followed to purify BgaX, the imidazole used for protein elution should be removed as quickly as possible to minimize loss of the enzyme through aggregation. The mass of BgaX, as determined from mass spectrometry, was 64849 Da, a value that is slightly higher than the expected mass of 64836 Da as predicted from the pri-mary amino acid sequence. However, N-terminal sequencing of this histidine-tagged protein was consistent with the predicted sequence (ATPESW...). The 13 Da differ-ence between the experimental and theoretical masses is most likely an artifact from cloning. The values of K m and kcat for the hydrolysis of pNPGal by the 6-histidine tagged BgaX at pH 7.0 (Km=53.5 / /M, kcat=87 s _ 1) were shown to be essentially the same as those for the non-tagged enzyme (Km=55.7 fjM, kcat=105 s - 1 ) 1 . It was therefore concluded that although the mass of the protein was slighter higher than expected, the incorporation of this tag did not affect the enzyme in any way that altered its activity. Hence, for the purposes of this report, 'BgaX' refers to the 6-histidine tagged wild type enzyme unless noted otherwise. 1 Karen Rupitz, unpublished data. Kinetics were done with 90-95% purified enzyme as determined by silver stained SDS-PAGE. C H A P T E R 2 The Characterization of X. manihotis /3-Galactosidase 22 2.2.2 pH Stability The stability of BgaX with respect to pH was determined by the measurement of residual enzyme activity after a 2 minute incubation at pH values ranging from 4-8 (Figure 2.4). A two minute incubation period was chosen since this was the typical assay time. From the results depicted in Figure 2.4, it can be seen that the enzyme is most stable at a pH of 6.0 or greater and activity is rapidly lost at lower pH values. 2.2.3 pH Dependence The dependence of k c a t and k c a 4 / K m of BgaX on pH was determined using DNPGal as a substrate (Figure 2.5). The bell shape of these plots indicates that both kinetic parameters are dependent on two ionizations of the enzyme. The upper pK a value of each plot is deemed as more reliable as compared to that of the lower pK a value due to the limited data that were obtained below pH 5.0 as the result of the rapid loo-se -l y -(—i •§ 60-o < 40-20-4 5 6 7 8 pH F I G U R E 2.4: The stability of BgaX with respect to pH. The curve shown is for illustrative purposes only. C H A P T E R 2 The Characterization of X. manihotis B-Galactosidase 23 (a) (b) I 1 1 1 1 1 I 1 1 1 r F I G U R E 2 .5 : T h e d e p e n d e n c e of (a) kcat/Km a n d (b) kcat of B g a X o n p H . B o t h d a t a sets were fit to y = 1 0 ( 2 » p t f _ p K a i - p ( C 2 ) + ^ o ( p ^ - p ^ a i ) + i ( s e e M a t e r i a l s a n d M e t h o d s ) . inactivation of BgaX at these pH values. The first irreversible step of catalysis (glycosylation) is reflected by kcat/Km and is dependent on the protonation state of both active site carboxylates (Figure 2.5 (a)). The lower pK a of 4.9 ± 0.2 is therefore attributed to the catalytic nucleophile, and the upper value of 6.4 ± 0.2 to the general acid catalyst. The rate determining step for the hydrolysis of DNPGal by BgaX is deglycosylation (vide infra) and is therefore dependent on the protonation state of the general base catalyst. Therefore the lower ionization of the kcat versus pH plot (Figure 2.5 (b)) with a pK a of 3.7 ± 0.3 is presumably representative of this catalytic residue. It is illustrated in Figure 1.2 that a single carboxyl group functions as both the general acid and the general base during catalysis. The values extrapolated from Figure 2.5 indicates that the pK a of this residue changes ca. 2.7 units during catalysis, presumably due to local electrical changes within the enzyme active site as the result of the removal of charge from the catalytic nucleophile upon forming the covalent C H A P T E R 2 The Characterization of X. manihotis (3-Galactosidase 24 glycosyl enzyme intermediate. Similar shifts of the p K a of this residue have been observed for Agrobacterium sp. glucosidase (Abg) [10] and Bacillus circulans xylanase (Bex) [37]. The origin of the dependence of kcat on a moiety with a p K a of 7.1 ± 0.1 is not clear. This may be the result of dependence on the side chain of another amino acid residue close to or in the enzyme active, site that is involved in crucial hydrogen bonds or similar non-covalent interactions during catalysis. The activity of Abg with a deg-lycosylation rate limiting substrate has also shown to be dependent on an ionization with a p K a of ca. 8.0 [10], suggesting the participation of a mechanistically important residue similar to that for BgaX. It is possible that the upper p K a dependence of the kcat versus pH plots of these enzymes is attributed to the ionization of a tyrosine residue which hydrogen bonds to the catalytic nucleophile, as was shown to occur in Bex through crystallographic studies [38]. All subsequent analyses of BgaX were done at pH 6.0 in order to maximize enzy-matic activity while maintaining protein integrity. 2.2.4 H y d r o l y s i s k i n e t i c s Kinetic parameters for the hydrolysis of several pNP-glycosides by BgaX are listed in Table 2.1. With the exception of pNPGal, the kinetic analysis of all pNP-glycosides was limited by substrate solubility, making it impossible to measure hydrolysis rates at concentrations significantly higher than K m . However, the measured initial velocities corresponding to the highest possible substrate concentrations appeared to approach a maximum, enabling an adequate fit of equation 2.1 and extrapolation of realistic values of K m and kcat. Yet, since enzyme saturation was only definitively achieved with pNPGal, the kinetic parameters determined for all other pNP-glycosides are C H A P T E R 2 The Characterization of X. manihotis (3-Galactosidase 25 T A B L E 2.1: Kinetic parameters for the hydrolysis of a range of nitrophenyl-glycosides by BgaX. kcat/Km A AG** Substrate Km(mM) &cat(s-1) ( s _ 1 r a M _ 1 ) (kJ(kcal)/mol) DNPGal 0.070 ± 0.014 36 ± 2 600 ± 100 0.06 (0 pNPGal 0.050 ± 0.002 35.8 ± 0.3 700 ± 30 pNPFuc 3.5 ± 0.3 7.2 ± 0.3 2.1 ± 0.2 15 (3.6) pNP-a-L-Ara 1.3 ± 0.4 0.34 ± 0.05 0.27 ± 0.08 20 (4.8) pNPGalNAc 0.71 ± 0.14 0.0217 ± 0.0012 0.030 ± 0.006 26 (6.2) pNPGlc 2.9 ± 0.5 0.080 ± 0.004 0.027 ± 0.005 26 (6.3) pNPXyl 4.3 ± 1.4 0.050 ± 0.006 0.012 ± 0.004 28 (6.8) M4) * A A G * = AG^pNP-glycoside) - AG*(pNPGal); values of AG* were calculated with Equation 2.4 F I G U R E 2.6: Structures of the pNP-substrates of Table 2.1. Shaded areas indi-cate how each glycoside differs from pNPGal. C H A P T E R 2 The Characterization of X. manihotis B-Galactosidase 26 viewed as approximate values. The rate determining step of catalysis with D N P - and pNPGal There are two steps associated with the catalytic mechanism of hydrolysis by BgaX: glycosylation and deglycosylation (Figure 1.2). The determination of which step is rate limiting for a particular substrate can be achieved by comparison of the kcat values for different nitrophenyl derivatives. The p K a values of the activated leaving groups of DNP- and pNPGal differ by 3.4 units (pKf NP= 3.8, yK*?p= 7.2)2. Table 2.1 reveals that kcat is essentially the same for DNP- and pNPGal, indicating that the leaving group ability of the aglycone has no effect on the rate of hydrolysis. This could only be true if glycosylation is not rate limiting; since this step involves the displacement of the aglycone, the rate should be dependent on its p K a . The rate determining step for the hydrolysis of DNP- and pNPGal with BgaX is therefore most likely deglycosylation, but factors such as the initial association of the substrate with the enzyme, and conformational changes of the protein during catalysis may also play significant roles. A more detailed investigation involving kinetic studies with a series of substrates and measurement of kinetic isotope effects would, however, be needed to confirm this hypothesis. 2.2.5 Glycone specificity Although the rate determining step for the hydrolysis of DNP- and pNPGal by BgaX was examined, a similar study was not done for the remaining glycosides used as substrates (Figure 2.6) as their DNP- derivatives were not available. Since the rate determining step was not known for these remaining substrates, the comparison of kcat 2See Materials and Methods. C H A P T E R 2 The Characterization of X. manihotis (3-Galactosidase 27 values associated with the hydrolysis of each is of little use since this parameter may reflect different steps of catalysis in each case. Similarly, since kcat is a component of K m (Figure 2.1), the pseudo dissociation constant should also not be considered as an adequate value on which to base analyses. A better parameter by which to examine the glycone specificity of BgaX is kcat/Km since it reflects the overall efficiency of catalysis; that is, it is a measure of the energy barrier that exists between the free enzyme plus substrate and the transition state of the first irreversible step (AG*) . Values of A AG* (relative to AG* of pNPGal) were calculated with the use of kcat/Km and Equation 2.4 in order to examine the effect of changes in substrate structure on enzyme catalysis (Table 2.1). where k = Boltzmann's constant R = the molar gas constant h = Planck's constant and T = temperature There are two predominant factors with respect to the association between sub-strate and enzyme that contribute to the efficiency of catalysis: (i) electronic factors affect the formation and breakdown of the oxocarbonium ion-like transition state, and (ii) various non-covalent interactions between the enzyme and substrate directly af-fect binding. For the substrates used in the study of BgaX, it is difficult to determine the contribution of each of these effects arising from a change in substrate structure. Therefore, the calculated values of A A G * are representative of the combination of both electronic and binding factors that affect catalysis. C H A P T E R 2 The Characterization of X. manihotis 3-Galactosidase 28 The greatest values of A A G * were seen for pNPGlc and pNPXyl (AAG* = 26-28 kJ/mol, 6.3-6.8 kcal/mol). These results illustrate a determinant for the specificity of a glycosidase. That is, the epimerization at a single position has a very significant effect on the efficiency of catalysis. These effects are likely the result of the loss of stabilizing interactions between the enzyme active site and the epimerized alcohol, and the subsequent introduction of steric interference. The ability of BgaX to use pNPGlc and pNPXyl as substrates, albeit in a dimin-ished capacity, may reflect a distant ancestral relationship to a cellulase or xylanase as was previously postulated by Taron and co-workers based on amino acid sequence similarities (§1.4) [31]. It should be noted however, that it has been reported that BgaX is not capable of hydrolyzing terminal /3-(l—>4) linked glucose or xylose or /3-(l —»3) linked glucose from oligosaccharides [31], indicating that the activity of BgaX with pNPGlc and pNPXyl is likely greatly facilitated by the inclusion of a good leaving group. The comparison of A A G * associated with pNPGlc and pNPXyl with that of pNP-a-L-Ara illustrates the care with which kinetic data should be analysed. These substrates differ structurally only by the presence or absence of the hydroxymethyl at the 6-position, and epimerization at the 4-position (Figure 2.6). A preliminary analysis of the A A G * values calculated for pNPGlc and pNPXyl suggests that the epimerization at the 4-position contributes 26 kJ/mol (6.3 kcal/mol) to the reac-tion barrier, while elimination of the 6-position hydroxymethyl adds an additional 2 kJ/mol (0.5 kcal/mol). However, pNP-cv-L-Ara, which only lacks the hydrox-ymethyl at the 6-position as compared to galactose, has a A AG* value of 20 kJ/mol (4.8 kcal/mol). Therefore, in this particular study, the contributions of each discrete structural change within the substrate to the magnitude of the reaction barrier are not C H A P T E R 2 The Characterization of X. manihotis (3-Galactosidase 29 additive; indeed, they are somewhat compensatory. Values of A A G * must therefore be analysed in terms of alterations in substrate structure as a whole. The A A G * for pNPGalNAc was comparable to that for pNPGlc and pNPXyl (26 kJ/mol, 6.2 kcal/mol). It has been suggested that substitutions at the 2-position of a substrate are particularly detrimental to enzymatic hydrolysis due to confor-mational changes incurred during catalysis [8, 39]. Upon binding, the ground state chair conformation of the substrate is distorted into a half-chair to achieve the oxo-carbonium ion-like transition state. This conformational change requires the direct repositioning of the substituent at the 2-position. Therefore, any alterations made at this site would have an intimate effect on the formation of the required transition state. The substantial value of A A G * calculated for pNPGalNAc therefore most likely reflects this increased sensitivity of BgaX with respect to alterations at the 2-position of the substrate. The larger size of this substituent as compared to that of an hy-droxyl is presumably responsible for disfavoring the conformational alteration of the substrate within the enzyme's active site to impede the formation of the transition state. The lowest values for A AG* were seen for pNPFuc (15 k J/mol, 3.6 kcal/mol) and pNP-a-L-Ara (20 kJ/mol, 4.8 kcal/mol). The reduced catalytic efficiency ob-served with these substrates is most likely dominated by the loss of hydrogen bonding interactions between the 6-hydroxyl and the active site of BgaX. C H A P T E R 2 The Characterization of X. manihotis 8-Galactosidase 30 2.2.6 Inactivation by 2FDNPGal The activity of BgaX in the presence of 2FDNPGal decreased in a time-dependent fashion according to pseudo first order kinetics (Figure 2.7 (a)): Vn A(e-k°™^ +C (2-5) where and v/V0 kobs inact A,C the measured fraction of enzymatic activity at time t the observed rate of inactivation constants Saturation of BgaX with respect to inactivator was not achieved since, at high concentrations of 2FDNPGal, inactivation was too rapid to monitor reliably. However, values of Kj = 0.031 ± 0.005 mM and kinact = 0.030 ± 0.004s-1 were extrapolated 5 10 15 Time (min) 0.0 0.2 0.4 0.6 0.8 1.0 l/r2FDNPGall ( m M - 1 ) F I G U R E 2.7: (a) The time-dependent first order (Equation 2.5) decrease in ac-tivity of BgaX upon incubation with • 1.15 fiM, • 2.30 / / M , • 11.5 pJM, A 23.0 /uM, and • 46.0 u-M 2FDNPGal . (b) The double reciprocal plot ( f e ^ = [2FDNPGal] [l£t) + 3 E " b ) U S i n § t h e ktlct v a l u e S fr«m (a)-C H A P T E R 2 The Characterization of X. manihotis (3-Galactosidase 31 from the double reciprocal plot of Figure 2.7 (b). While these individual values are not too reliable, the ratio (kinact/Ki), which can be determined from the slope, is. The consequences of replacement of the alcohol at the 2-position by fluorine can be estimated by the comparison of the rate of glycosylation, reflected by kinact/Ki ( 1 . 0 ± 0 . 2 m M - V 1 ) and kcat/Km for DNPGal ( 5 0 0 ± 1 0 0 mM^s" 1 ) . This comparison illustrates that the fluorine substitution effectively reduces the rate of the first step of catalysis by 500-fold. Protection from inactivation The ga/acio-configured pyridoimidazole (galacto-imidazole) (Figure 2.8), an analog of nagstatin, a naturally occurring inhibitor isolated from Streptomyces amakusaensis [40], has been shown to be a good competitive inhibitor of /3-galactosidases from al-mond and E. coli (IC50 = 0.0016-0.10 /zg/mL) [41]. This inhibitor was demonstrated to also be a competitive inhibitor of BgaX. The Lineweaver-Burk plot of the initial reaction velocity at five different concentrations of pNPGal in the presence and ab-sence of galacto-imidazole is shown in Figure 2.9 (a). To within error (intercepts = 15 ± 4 with galacto-imidazole and 22 ± 7 for the control), the two lines intersect on the y-axis, a pattern characteristic of competitive inhibitors [42]. An approximate K$ value of 15 nM for galacto-imidazole with BgaX was calculated OH PH HO FIGURE 2.8: Galacto-conHgured pyridoimidazole (galacto-imidazole). C H A P T E R 2 The Characterization of X. manihotis B-Galactosidase 32 0.00 0.05 0.10 1 / p N P G a l ] O M ) F I G U R E 2.9: (a) Lineweaver-Burk plot of initial reaction velocity versus concen-tration of PNPGal, in the presence • and absence • of 7.2 nM galacto-imidazole, where slope; and slope0 are the respective slopes of each linear fit. (b) Fractional loss of activity of BgaX over time caused by 46.0 / i M 2FDNPGal in the presence • and absence • of 40 nM galacto-imidazole; data sets were fit to a first order expression (Equation 2.5). from the following relationship3 using the resultant slopes of the data of Figure 2.9 (a): [I] (2-6) slopei/' slope o — 1 Galacto-imidazole was shown to effectively protect BgaX from inactivation by 2FDNPGal . The fractional loss in activity of BgaX by 2FDNPGal in the presence and absence of galacto-imidazole (Figure 2.9 (b)) yielded a rate constant ratio [k°^s/k°bs) of 0.43, which is in close agreement with the value of 0.48 predicted by Equation 2.3. Since the rate of inactivation by 2FDNPGal decreased by the expected amount in the presence of a competitive inhibitor, the 2-fluoro-glycoside is presumably active-site directed. 3See Appendix A for derivation. C H A P T E R 2 The Characterization of X. manihotis f3-Galactosidase 33 Catalytic competance of the inactivated enzyme Once BgaX was inactivated by 2FDNPGal and the excess inhibitor removed, the enzyme spontaneously regained hydrolytic activity over time according to pseudo first order kinetics (Equation 2.7) with a measured rate constant (kreact) of 0.018 hr"1 (Figure 2.10). ^- = A{\ - e-kreactt) + C (2.7) 'o The observed spontaneous enzyme reactivation supports the belief that inactivated BgaX exists as a catalytically relevant species; that is, reactivation is attributed to the eventual turnover of the inhibitor-enzyme complex. Therefore, kreact can be described as the rate constant for the deglycosylation step of the 2-fluoro-glycosyl enzyme inter-mediate, and as such, can be compared to kcat for DNPGal since it too is related to Time (hr) F I G U R E 2.10: The spontaneous reactivation of 2-deoxy-2-fluorogalactosyl-BgaX. Data was fit to Equation 2.7. C H A P T E R 2 The Characterization of X. manihotis B-Galactosidase 34 this step of catalysis. The value for kreact is 8 x 106 times smaller than kcat, thereby illustrating the effect of the fluorine substitution at the 2-position on deglycosylation. The decrease in the rate of this step upon this substitution is significantly greater than that observed for glycosylation (500-fold). This difference is comparable to that documented for other glycosidases [17, 43, 44], and is presumably due to the greater leaving group ability of DNP as compared to that of the active site carboxylate of the glycosyl enzyme intermediate. The greater oxocarbonium ion-like character of the transition state of deglycosylation versus glycosylation [10] may also contribute to the increased sensitivity of deglycosylation with respect to the fluorine substitution. The catalytic competence of the inactivated enzyme species was also demonstrated by the increase in the relative rate of enzyme reactivation in the presence of an acceptor (k°rans) a s compared to kreact. The greater value for k°^ns indicates that reactivation was accelerated due to transglycosylation. These results are outlined in detail in Chapter 3. The observed protection by galacto-imidazole of BgaX from inactivation, and com-parison of the values of k°^ns and kreact, supports the belief that once inactivated by 2FDNPGal, BgaX exists as the 'trapped', yet catalytically competent fluoro-glycosyl enzyme intermediate. 2.2.7 The identification of the nucleophile of X. manihotis (3-galactosidase The expected increase in mass of BgaX if 2-deoxy-2-fluoro-galactose is covalently attached at the active site is 165 Da. The mass spectrum of inactivated wild type BgaX yielded an apparent mass of 65558 ± 5 Da, which was 171 Da greater than that of a control of non-inactivated enzyme. This mass difference suggests that the 2-C H A P T E R 2 The Characterization of X. manihotis B-Galactosidase 35 15 20 25 30 35 40 45 Time (min.) 15 20 25 30 35 40 45 Time (min.) 0 200 400 600 800 10001200 m/z 200 400 600 800 10001200 m/z F I G U R E 2.11: TIC of the control (a) and labelled (c) BgaX after peptic digestion. The mass spectrum of the peak at ca. 28 min. of the TIC for the control (b) and labelled (d) enzymes showing the m/z of the major species. fluoro-glycosyl enzyme intermediate is formed in a 1:1 stoichiometric reaction between BgaX and 2FDNPGal to form a relatively stable species, as anticipated. Digestion of inactivated histidine-tagged BgaX with pepsin resulted in numerous peptide fragments as shown in the total ion chromatogram (TIC) H P L C trace of Figure 2.11. The TIC of the digested inactivated enzyme was similar to that of the non-inactivated control except for the peaks with an elution time of ca. 28 min. The mass to charge ratio (m/z) profiles of the peptide ions in these fractions are similar except for two major signals: m/z = 869.0 in the control, and m/z = 951.0 in the inactivated samples (Figure 2.11 (b), (d)). The difference between the m/z values for these two peptides is 82.0 which is, C H A P T E R 2 The Characterization of X. manihotis f3-Galactosidase 36 within error, one half of the expected mass increase if 2-deoxy-2-fluoro-galactose is covalently linked to the enzyme (165 Da), suggesting that z = +2 for these peptides. Hence, 951.0 x 2 - 2 H + = 1900.0 Da and 869.0 X2 -2H+ = 1736.0 Da were the masses of the 'labelled' and control peptides originating from the inactivated and control enzymes respectively. Therefore, the peptide sequence that bracketed the catalytic nucleophile had a mass of 1736 Da. A search of the primary amino acid sequence of BgaX for a peptide with this mass showed that there are two candidate sequences that contain the putative nucleophile, G lu 2 6 0 (underlined): 2 4 8 I K F R P D Q R P M V G E Y 2 6 1 and 2 5 8 V G E Y W A G W F D H W G K 2 7 1 . Both of these sequences contain a single Asp and Glu residue, either of which could potentially act as the nucleophile, based on the conservation of such catalytic residues in other glycosyl hydrolases. The precise identity of the position of the nucleophile in BgaX was achieved through further analysis of the m/z = 951.0 and 869.0 ions of the labelled and control peptides respectively in the daughter ion scan mode (MS/MS). These par-ent peptides were each separately fragmented further into two separate segments at three distinct peptide bonds (Figure 2.12). Two of the three daughter fragments of these parent peptides that were cleaved at the carboxyl end (B n ions) were identi-fied in both the labelled and control samples as 2 4 8 I K F R 2 5 1 (B 4, m/z = 545.0) and 2 4 8 I K F R P D 2 5 3 (B 6, m/z = 757.0). These two daughter ions originating from the la-belled and control species had the same m/z value, indicating that Asp 2 5 3 was not attached to the 2F-galactose moiety, and hence, does not function as the catalytic nucleophile. Three daughter ions that were produced by cleavage of the labelled parent peptide at the amino end (yl^belled^ were larger than fragments cleaved at the same site in the control peptide (Ycnontro1) by 164.0 ± 0.5: 2 5 2 P D Q P R M V G E Y 2 6 1 ^labelled m / z = 1 355.5 ; Ycontrol m ^ = U 9 1 5 ) ; 2 5 4 Q P R M V G E Y 2 6 1 (Yl*belled m/z = C H A P T E R 2 The Characterization of X. manihotis 3-Galactosidase 37 (a) (b) 1 2 -1 0 -o X 8 -6 -H—' <u 4 -2 -0 -o x c 8 6 9 . 0 B 4 5 4 5 . 0 7 5 7 . 0 Y 7 851 6 0 0 8 0 0 Y 6 9 8 0 . 0 Y 4 1191.5 6 5 -4 -3 2 1 0 1000 (m/z) I 1200 951.0 B 4 5 4 5 . 0 757 6 .0 jljL.ii.il uiiiJ i, Ji . i i 1144.5 o X 1400 Y 7 Y 4 1015.0 1355.5 ^ i l l I M i l l I 6 0 0 8 0 0 _ j i J _ 1000 ( m / z ) 1200 1400 (c) A n C n CH-R, - C C r - N H -B 4 B 6 By 248 CH I—K—F—R-R n+1 Xn Yn Z n -P—D- - Q - - P — R — M — V — G — E — Y 2 6 1 Y 4 Y 6 Y 7 F I G U R E 2.12: MS/MS of the parent (a) m/z = 869.0 peptide of the control, and (b) m/z = 951.0 peptide of the labelled species, (c) Fragmentation patterns of peptides in general and specifically for the parent peptides from BgaX. C H A P T E R 2 The Characterization of X. manihotis B-Galactosidase 38 1144.5; Yfntro1 m/z = 980.0), and 2 5 5 P R M V G E Y 2 6 1 (Yl7abelled m/z = 1015.0; ycontroi M / Z _ 3514) Therefore, the amino acid labelled with the 2-fluoro-galactose moiety was contained within residues 255-261. As G l u 2 6 0 is the only residue with a carboxylic acid side chain in this sequence, it was concluded that this amino acid functioned as the catalytic nucleophile of BgaX. 2.2.8 The nucleophile mutant E260A BgaX The mutant E260A BgaX was constructed through direct gene manipulation and was overexpressed in E. coli. The enzyme was purified to ca. 90% purity as estimated by silver stained SDS-PAGE. The BgaX mutant lacked hydrolytic activity, as was expected since the anticipated nucleophilic carboxyl of glutamic acid was replaced with the non-nucleophilic methyl side chain of alanine. Although E260A BgaX did not hydrolyze nitrophenyl glycoside substrates on its own, activity with DNPGal was recovered by the provision of azide. This finding indicates that the active site architecture remains intact in the mutant enzyme to productively bind the substrate. The addition of azide presumably rescues activity by taking the role of the mutated nucleophile of BgaX as shown in Figure 2.13. */^/v^/>/^/^/\/, s/VV'yrv/V"' c r o-*^ cr ^ o e ?HPH A N o 2 OHOH ^ H O V ^ l HO _ OH O H 3 0 I H © C H 3 DNP C K F I G U R E 2.13: Azide rescue of the activity of E260A BgaX C H A P T E R 2 The Characterization of X. manihotis B-Galactosidase 39 Although a thorough analysis of E260A BgaX was not undertaken, the observation that activity was recovered in the presence of azide supports the identification of G l u 2 6 0 as the catalytic nucleophile of BgaX. 2.3 Conclusions The brief study of the glycone specificity of BgaX illustrates the effect that small changes in substrate structure have on enzymatic activity. Each alcohol group in the substrate influences the productive association with the enzyme to contribute to substrate specificity and the overall efficiency of catalysis. Further, the ability of BgaX to hydrolyze pNPGlc and pNPXyl , coupled with the homology of a region of this enzyme with that of family 10 cellulases and xylanases (Figure 1.10), suggests that this /3-galactosidase may have evolved from the shuffling of discrete domains from ancestral glycanases. The glycoside 2FDNPGal was demonstrated to be an active site-directed, mechan-ism-based inactivator of BgaX. The substitution of fluorine at the 2-position of DNP-Gal decreased the rates of both steps of catalysis, with the rate of deglycosylation decreasing several orders of magnitude more compared to that of glycosylation. The net .effect of the activity of BgaX with 2FDNPGal was a trapped, yet catalytically competent 2-fluoro-glycosyl enzyme intermediate with a half life of ca. 40 hr. The longevity of this intermediate allowed its analysis by mass spectrometry both in the intact and digested enzyme to permit the elucidation of G l u 2 6 0 as the active site nu-cleophile of BgaX. The assignment of this catalytic residue was further confirmed by the construction of the E260A BgaX mutant and the rescuing of its activity in the presence of azide. Future studies should fully investigate the capability of this and C H A P T E R 2 The Characterization of X. manihotis B-Galactosidase 40 other nucleophile mutants of BgaX (eg. serine) to function as a glycosynthase. This finding confirms the previous prediction of the location of this active site residue that was based on primary sequence similarities with other members of family 35, and agrees with the assignment of the nucleophile of the human /5-galactosidase. Therefore, adequate comparisons can be made between such diversely related organ-isms as human and X. manihotis in order to identify catalytically important residues, provided of course that significant sequence similarities exist elsewhere in the enzymes. The aforementioned weak sequence homology among members of glycosyl hydrolase family 35 in the vicinity of the catalytic nucleophile is interesting given the importance of this active site residue. Studies that would further investigate how enzymes from such a diverse collection of organisms could be grouped in the same family would be of interest. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 3.1 Introduction 3.1.1 Screening of potential acceptors It was noted previously (§1.3.1) that in order to maximize yields of transglycosy-lation reactions, it is desirable to be able to probe the specificity of the aglycone site of a glycosidase to identify the most suitable acceptors for the particular wild type enzyme or glycosynthase. This could be accomplished by the screening of a number of compounds to determine their degree of favorable association with the aglycone site. We have developed such a procedure with the use of 2-deoxy-2-fluoro-glycosides that is simple and has the capacity to rapidly screen a large number of potential acceptors. A glycosidase inhibited by a 2-deoxy-2-fluoro-glycoside will eventually be reacti-vated through the nucleophilic attack on the trapped glycosyl enzyme intermediate by water or an acceptor. It has been previously demonstrated that the rate of such reactivation varies in the presence of different acceptors. The 2-fluoro-glucosyl en-zyme species of Agrobacterium sp. /3-glucosidase (Abg) was previously demonstrated 41 C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 42 in this laboratory to turn over 2700-fold faster in the presence of pNP /?-glucoside as compared to buffer alone, and only 600-fold faster in the presence of cellobiose [12]. Since the trapped fluoro-glycosyl-enzyme species very closely resembles the same in-termediate formed in a regular transglycosylation reaction, the influence an acceptor has on the rate of enzyme reactivation should be indicative of the specificity of the aglycone site with respect to that particular acceptor. That is, the greater the de-gree of productive binding of the acceptor with the aglycone site, the greater the rate of reactivation due to transglycosylation. This is the basic principle of the de-veloped screening methodology: the specificity of the aglycone site of a glycosidase with respect to transglycosylation can be examined by comparison of the rates of reactivation of the inhibited enzyme in the presence of various potential acceptors. Once the most suitable acceptors for an enzyme are determined, they can be used with non-fluorinated donors for transglycosylation reactions. By identifying the best acceptors for a glycosidase, the most appropriate enzyme can be chosen as a catalyst for a desired reaction. The basis of this screening methodology was briefly examined by D. Stoll in col-laboration with this laboratory [28]. He compared the rates of reactivation of Cel-lulomonas fimi /3-mannosidase Man2A that had previously been inactivated with 2-deoxy-2-fluoro-/3-mannosyl fluoride in the presence of nine glycosides. It was found that of these potential acceptors, only four induced a significant reactivation rate rel-ative to a control in buffer alone. It is evident from this acceptor 'hit' rate of ca. 50% that in order to fully examine aglycone specificity, it is desirable to screen a large number of compounds. Furthermore, there are vast numbers of compounds that one may wish to use as acceptors, even if only oligosaccharides are taken into consider-ation. However, virtually any compound that requires glycosylation can be viewed C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 43 as a potential acceptor for a transglycosylation reaction. For both of these reasons, it was desirable to be able to evaluate the aglycone specificity of a glycosidase in a high throughput screen. The development of 96-well plate readers for kinetic analyses has made this a relatively simple task. A glycosidase inhibited by a 2-deoxy-2-fluoro-glycoside is added to each well of a 96-well plate that contains a different potential acceptor to be screened. After incubation for a period of time, each well is assayed for enzyme activity arising from reactivation by the addition of a nitrophenyl-glycoside substrate. Those wells that show greater enzyme activity relative to a control in-cubated in buffer alone indicate a greater reactivation rate, and thus contain those compounds that would be the best candidates to act as acceptors in a transglycosy-lation reaction. Once these 'positive hits' were identified, we desired a more detailed look at the kinetics of enzyme reactivation in the presence of each positively screened compound. This was accomplished by the same strategy described above for the 96-well plate screening process. In this instance however, aliquots are removed from each reactiva-tion mixture over time and individually assayed for enzyme activity. In this way, the effect of each positive hit on enzyme reactivation could be more accurately determined. 3.1.2. Preparative-scale transglycosylation reactions Once the described acceptor screening methodology was applied to a glycosidase, it was necessary to demonstrate the utility of the identified positive hits with respect to functioning as acceptors in transglycosylation reactions. Four of the seven enzymes that were used in the screening process were used as catalysts in transglycosylation reactions with one of the identified positive hits acting as an acceptor in each case. The reactions were performed on a preparative scale and the resultant transglycosylation C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 44 products isolated and characterized and compared to other such products from the literature. The characterization of the transglycosylation products involves the determination of two parameters: (i) the number and identity of monosaccharide units; and (ii) the nature of the glycosidic linkage between these units. The first parameter can be easily determined via 1 H NMR, as the types of possible monosaccharides in the product are known, and there are numerous spectral data in the literature available for comparison and subsequent identification. The determination of the linkage between saccharide units is not as easily deter-mined. It was mentioned briefly in §1.3.1 that the precise mode of interaction between the aglycone site of a glycosidase and the acceptor in a transglycosylation reaction will determine which hydroxyl of the acceptor is in a position to attack the glycosyl-enzyme intermediate. Depending on the positioning of the acceptor in the aglycone site, a glycosidic bond may be formed in the transglycosylation product that does not correspond to the preferred linkage of the enzyme with respect to hydrolysis. There is also the possibility of a glycosidase catalyzing the formation of several different linkages in a single transglycosylation reaction. This phenomenon has been demon-strated for a number of enzymes, and again, is linked to the particular acceptor used in each reaction [12, 20, 28, 45]. The developed acceptor screening strategy identifies those acceptors that are best suited for a particular glycosidase for transglycosylation reactions. It does not how-ever predict the linkages that will be formed when such acceptors are used in actual reactions. It was therefore necessary to identify the types of glycosidic bonds formed in the preparative-scale transglycosylation products by conventional means. Two of the most common ways in which to identify the linkage between saccha-C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 45 ride units involve the comparison of HPLC retention times with those of standards, or methylation analysis. If a transglycosylation reaction is monitored by HPLC, the formation of the products is readily apparent. However, in order to identify a product in this manner, it is necessary to have a standard sample of the product in question in order to make comparisons between column retention times. While it may be possible to have access to a variety of such compounds, the question of regiochemistry with re-spect to transglycosylation means that any one reaction has the potential to result in the formation of a number of isomers. It would be very time consuming to chemically synthesize all potential products for their use as standard compounds. In methylation analysis, the linkage type is identified by methylating all free hydroxyls in the oligosac-charide in question and subsequent acid hydrolysis. The non-anomeric methyl ethers are stable to this hydrolysis, whereas glycosidic bonds are readily cleaved, leading to methylated monosaccharides that specifically reflect the linkage and sugar units of the parent oligosaccharide. Analysis of these resultant fragments is done by HPLC or GC. However, as in the case with straight HPLC analysis of transglycosylation reactions, it is necessary to have access to a large number of standards with which to make comparisons for identifications. If the necessary standards were available for this study, either of these strategies to identify the glycosidic linkages in the synthesized transglycosylation products may have been suitable. Yet the lack of such standards necessitated an alternative method. Over the past decade, developments in carbohydrate NMR have made it possible to determine the linkage types in an oligosaccharide through non-destructive means without the explicit need for standards with which to make comparisons. Spectra are commonly taken of the peracetylated oligosaccharide in a deuterated organic solvent, which results in narrower, more resolved signals as compared to the non-derivatized C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 46 sample in aqueous solution [46]. The following discussion refers to oligosaccharides that have been derivatized in this manner. A common method to determine the site of a glycosidic bond is to compare the signals of each ring proton of a peracetylated monosaccharide contained within an oligosaccharide to that of the isolated unit. Glycosylation of a sugar residue typically shifts the chemical signal of the proton at the linkage site by -0.2 to -0.26 ppm, while the rest of the protons are relatively unaffected [47]. These 'glycosylation shifts' can thus be used to determine the site of a glycosidic bond, provided that several substan-tial proton shifts are not observed, as is often the case. In the case that glycosylation shifts do not provide adequate insight into linkage types in oligosaccharides, 2D NMR experiments are often of great help. Heteronuclear multiple bond correlation (HMBC) NMR experiments identify 3- and 4- bond correlations within compounds, a sensitiv-ity that is sufficient to trace the connectivity of two sugars via coupling between ring protons at the glycosidic bond. Recently a simple, yet powerful, methodology to elucidate linkage sites in carbo-hydrates has been developed that is based on 1 H - 1 3 C couplings [48, 49, 50, 51]. The natural abundance of 1 3 C is too low to normally detect any such couplings between ring protons and the carbonyl carbon of the adjacent acetyl group. However, the use of an isotopically enriched reagent to derivatize the oligosaccharide, such as 1-13C-acetyl chloride, enables the detection of a J-coupling between the ring protons and their adjacent carbonyl carbons. These additional splittings are in the range of 2.5-4.7 Hz and are thus distinguishable in 1D-1H spectra [51]. Protons that were not initially adjacent to an hydroxyl in the non-derivatived sugar, such as the anomeric proton of the non-reducing end monosaccharide and those adjacent to glycosidic linkages, do not experience this additional splitting. Therefore, the connectivity between carbo-C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 47 hydrate units can be elucidated by the determination of which proton signals did not experience an additional splitting from the 13C-enriched acetyl groups. The methods of glycosylation shift, and derivatization with 13C-enriched acetyl groups were used to determine the glycosidic linkages in the transglycosylation prod-ucts synthesized in this study. 3.2 Results and discussion 3.2.1 Large scale screening of potential transglycosylation ac-ceptors The described transglycosylation acceptor screening methodology was applied to seven glycosidases: BgaX, Bacillus circulans /3-galactosidase (BgaC), Agrobacterium sp. /3-glucosidase (Abg), Cellulomonas fimi /5-mannosidase (Man2A), Streptomyces lividans endoglucanase (CelB), Cellulomonas fimi xylanase/glucanase (Cex), and hu-man /3-glucuronidase (HBG). The complete list of carbohydrates that were screened and the positive hits associated with each are provided in Tables 3.1 and 3.2. The mono- and disaccharides that were chosen were selected for several reasons: they are representative of carbohydrates that comprise many common synthetic targets; they are interrelated by simple substitutions or epimerizations, thereby illustrating the ef-fect of subtle structural changes in the acceptor on aglycone binding; and they were readily available in the laboratory. Compounds were listed as a positive hit if they induced a higher degree of enzyme reactivation at the time of assaying as compared to that of a control in buffer alone. However, some of those glycosides screened induced a lower amount of enzyme reac-C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 4 8 tivation with respect to a control. Two of the possible explanations as to why this may occur follow: (i) Since the K m of each potential acceptor with the aglycone site of each enzyme was not known, the inactivated glycosidases were incubated with a high concentration of the glycoside to be tested (~30 mM or 60% saturation)1. Upon measurement of enzymatic activity to determine the amount of 2-fluoro-glycosyl enzyme intermediate that had turned over, it is possible that reactivated enzyme was inhibited to some degree by the excess of acceptor present. This inhibition could make it appear as though less enzyme had been reactivated as compared to a control. A simple way by which to test this theory would be to perform a control using non-inactivated enzyme. After incubation, a decrease in enzyme activity would identify those potential acceptors that inhibit the enzyme in question. (ii) The turnover of the 2-fluoro-glycosyl enzyme species may appear to be slower in the presence of a potential acceptor compared to that of a control due to the re-inactivation of the enzyme by the synthesized transglycosylation product. If the glycosidase in question has a high affinity for the transglycosylation product, it is possible that this product will not diffuse out of the enzyme's active site, but will re-main bound. The reactivated glycosidase may then remain inhibited in a competitive manner by the transglycosylation product. Alternatively, the enzyme may cleave the newly formed glycosidic bond to again inactivate the enzyme as shown in Figure 3.1. Whether points (i) and/or (ii) hold true, the observation that the incubation of a potential acceptor with a 2-fluoro-glycosyl enzyme species decreases the rate of en-1 Stock solutions of « 50 mM in water were made for all acceptors; some of these solutions were saturated. Dilution of these stocks for the screening procedure resulted in final acceptor concentra-tions of w 30 mM or 60% saturation. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 49 J W W V A A ' - ' J V W W W . / W W W V o^o0 0 A 0 . o^oQ j |J H O R ^ OHOH \ OH PH H j H O \ ^ r A _ y Reactivation H O * ~ * ^ Re-inactivation H O \ ^ \ o? ^ o e°\^ ° °- °^ F I G U R E 3 . 1 : R e a c t i v a t i o n of a g lycos ida se a n d subsequen t r e - i n a c t i v a t i o n b y i t s t r a n s g l y c o s y l a t i o n p r o d u c t . zyme reactivation is a clear indication that this compound would not be among the best candidates to act as acceptors in a regular transglycosylation reaction. If the presence of the acceptor inhibits the glycosidase, the transglycosylation pathway will be blocked. Also, if the enzyme in question has a high affinity for the transglycosy-lation product it may be inhibited by active site competition, or the newly formed glycosidic bond may be hydrolyzed, lowering the overall product yield. It should be stressed that for the purposes of developing the screening procedure only those compounds that would act as the best acceptors for a particular glycosidase were considered. Therefore, those potential acceptors that induced a degree of reactivation that was less than that seen for the control were treated as negative results and were not analyzed further. The total number of positive hits for each glycosidase (Table 3.1) were relatively low (7-16 out of a possible 45). Several general trends were seen with these positively screened carbohydrates: (i) Of the potential acceptors screened, aryl glycosides were generally better re-activators than their parent carbohydrates. This was evident through observations C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 50 T A B L E 3.1: Positive hits from screened glycosides. P O T E N T I A L A C C E P T O R S ' 1 G L Y C O S I D A S E BgaX BgaC Abg Man2A Cex CelB H B G arabinose fructose galactose pNP /?-galactoside + + + + 2-deoxygalactose ./V-Ac-galactosamine glucose pNP /?-glucoside + + + + + + + Ph /J-glucoside pAcPh ,9-glucoside D-glucal 1-cyanoglucal + + + + + + + + + + + + iV-Ac-glucosamine iV-Ac-glucalamine Ar-Ac-glucosamine-/3-NH2 pNPGlcA + + + + + 2-deoxyglucose pNP /3-mannoside pNP-a-xyloside pNP /3-xyloside + + + + + + + + + + + oNP /?-xyloside Me /3-xyloside xylose pNP /?-cellobioside + + + + + + + DNP ,5-cellobioside Me a-cellobioside Me /?-cellobioside lactose + + + + + + + + + + + pNP ,3-lactoside maltotriose gentiobiose Gal-/3-(l-3)-Glc-/9-SPh + + + + + + + Gal-0-(l-4)-Glc-0-SPh DNP /3-fucoside 2,5-DNP /3-xylobioside • + + + + "All glycosides have the D- configuration unless noted otherwise; see the List of Abbreviations for full carbohydrate names. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 51 T A B L E 3.2: Glycosides that are negative hits for all enzymes. Glycoside pNP a-L-arabinoside a-L-fucose oNP /?-galactoside D-galactal D-galacturonic acid acetyl /3-glucoside mannose pNP a-mannoside ./V-Ac-mannosamine such as pNP 8—galactoside being a positive hit for BgaC and Abg, yet galactose was not. Similar trends were seen for aryl glucosides and aryl xylosides; these carbohy-drates were positively screened with BgaX, BgaC, and Abg, whereas negative results were obtained for all three enzymes with the parent compounds glucose and xylose. This general trend was seen previously in this laboratory for the reactivation of Abg and Man2A. The turnover of the 2-fluoro-glucosyl enzyme species of Abg'increased 2700-fold in the presence of pNP /3-glucoside, yet an increase in reactivation rate was not seen with glucose [12]. Similarly, the reactivation of Man2A was significantly increased relative to a control in the presence of pNP /3-mannoside, but not with the parent compound mannose [28]. This finding is most likely the result of favorable interactions between the aryl substituent and the enzymes' aglycone site. One excep-tion to this trend was seen with HBG, which showed positive results for galactose and xylose, but not for any of their aryl derivatives, suggesting that similar interactions are disfavoured in the aglycone site of this enzyme. The finding that in general, aryl glycosides are better enzyme reactivators, and hence most likely the best candidates to act as acceptors, is especially useful from a synthetic viewpoint. If a nitrophenyl glycoside is used as an acceptor in a transgly-C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 52 cosylation reaction, the product will necessarily have a nitrophenyl substituent at its reducing end. This provides an activated center for further chemistry if the desired target requires additional synthetic steps. (ii) Although aryl glycosides were generally preferred as acceptors by compari-son with their parent carbohydrates, the reactivation of some enzymes was sensitive to the substitution of the phenyl substituent. It appears that para-nitro substituted phenyl glycosides were generally better reactivators relative to their ortho- derivatives: pNP /3-galactoside and pNP /3-xyloside were positive hits for BgaC and Abg, yet oNP /5-galactoside and oNP /3-xyloside were not. Similarly, BgaX accepted the para-substituted nitrophenyl /3-xyloside, but not the ort/io-substituted derivative. These examples illustrate the significant effect that small changes in acceptor structure have on productive binding to the aglycone site of the glycosidases studied. If an activated glycoside is to be used as a transglycosylation acceptor, it would be beneficial to de-termine which activated group would be best accommodated in the enzyme's aglycone site to ensure maximal product yields. Again, however, an exception to this general trend was seen with Man2A, which had positive hits with both p- and o-NP /3-xyloside, as well as with p-NP o-xyloside. Thus, Man2A either has a high tolerance with respect to substitution on the phenyl moiety, or associates with xylose in such a productive manner as to overcome any unfavorable interactions that may be incurred by an ortho- substituted aryl group or a change in the anomeric configuration of the acceptor. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 53 3.2.2 The kinetics of enzyme reactivation via transglycosylation Those compounds that were identified as positive hits in the large scale screen were studied further to determine the effect of their presence on the kinetics of enzyme reactivation. The fractional gain in activity of the 2-fluoroglycosyl-enzyme species with each potential acceptor over time (Figure 3.2) followed the same first order rate profile (Eqn. 3.1) seen for spontaneous reactivation (Figure 2.10). . v/V0 = A ( l - e - k ^ l ) + C (3.1) where V/V0 — the fractional gain in enzyme activity at time t Kllct = the observed rate of reactivation due to transglycosylation and C and A = constants It is important to note that when comparing first order reactivation profiles related to different acceptors for a given glycosidase, there are two dominant parameters: both k°besact and the upper limit of reactivation (A). The rationale behind the inadequacy of considering only k°besact when ranking acceptors is illustrated by the reactivation profiles of BgaX depicted in Figure 3.2. In the presence of pNPGal the reactivation rate of BgaX is slightly greater than that for pAcPhGlc (k°besact = 0.072 hr" 1 and 0.068 hr"1 respectively). However, from the examination of enzyme reactivation in the presence of these glycosides, it is readily apparent that the upper limit of reactivation for pNPGal is substantially less than that for pAcPhGlc. That is, in the presence of pNPGal, much less BgaX overall appears to be reactivated. There are several possible explanations as to why this may C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 54 T i m e [ h r ] F I G U R E 3.2: The reactivation of inactivated BgaX in the presence of • pAcPhGlc and O pNPGal. occur: (i) Over the course of the experiment the excess acceptor may be hydrolyzed by enzyme that has been reactivated. It was discussed in the previous section that aryl glycosides were significantly better acceptors than their parent glycosides. Therefore, if such an aryl glycoside is hydrolyzed, the produced sugar would not be as good an acceptor. Therefore, the concentration of 'good' acceptor is constantly decreasing during the experiment. While this result is obvious for such acceptors as pNPGal with BgaX, this scenario may also be applicable in other cases where the hydrolytic activity of the enzyme under study was not tested with the potential acceptors. There-fore, there is one drawback to this screening strategy: it may not reliably identify a glycoside as an acceptor if it is a substrate for the enzyme. It would therefore be beneficial to know if the potential acceptors to be screened are substrates to avoid false negative results. Interestingly, this shortcoming does not apply to all substrates for a particular enzyme, as is demonstrated by pNPGal, pNPXyl and pNPMan be-C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 55 ing identified as positive hits for BgaC, Abg and Man2A respectively. This apparent discrepancy is most likely a consequence of a higher rate of reactivation versus hy-drolysis at high concentrations of substrate. For BgaC and Abg, at concentrations of substrate that are several times K m (ca. x 8) the initial reaction velocity versus substrate curve deviates from the classical Michaelis-Menten expression, an indication that transglycosylation is occurring [10, 52]. The reactivation profiles were generated in the presence of 30 mM of acceptor, a concentration that would have been sufficient for substantial transglycosylation (reactivation) to occur versus hydrolysis. (ii) As was noted in the previous section, the newly formed transglycosylation product may be a good inhibitor of the enzyme under study. The transglycosylation product may be re-bound by the newly reactivated enzyme to again re-inactivate the glycosidase. This would make it appear as though less enzyme overall was reactivated. Therefore, there are several reasons why the upper limit of reactivation may differ between reactivation profiles involving different acceptors. It is therefore inadequate to directly contrast values of k°bsact to make comparisons between the compounds screened since this paramter may reflect additional steps aside from reactivation. In order to circumvent this problem, the initial velocity of each reactivation at t = 0 (Vgeact) was calculated from each reactivation profile as the product of the upper limit of reactivation and k°besact2. Several reactivation profiles deviated from a first order expression in that after increasing over time, instead of reaching a limit, it decreases, in some cases quite dramatically (Figure 3.3). As the observed deviation occurs after several hours, it could be the result of the newly synthesized transglycosylation product inhibiting the reactivated enzyme. Further experimentation is required however to confirm this 2 See Appendix A for the full derivation of V o r e Q C t . C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 56 0 1 2 3 4 5 Time [hr] F I G U R E 3.3: The reactivation profile of Man2A with pNP /9-xyloside. hypothesis. Although the transglycosylation products associated with reactivation profiles such as that depicted in Figure 3.3 may inhibit the enzyme after a significant amount is synthesized, the glycosides associated with these profiles were nonetheless considered to be good acceptors. In these cases then, a first order expression was fit to the lower portion of the reactivation profile, and the extrapolated value of VJeact ranked among the other 'well behaved' acceptors. For this reason, there is a substantial amount of error associated with some of the values of Vg6act. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 5 7 0) PH PH in CO OT 1 1 o Qa. o 1 1 d o o o O 1 1 S "N CO 1 1 PH 1 1 T 1 "cO cd o O £ ° i ° 1 d I Qa. 43 PU o < I ft tu G 6 cd OT o o d M I o < CD OT O CJ d >, X o I OT o CJ d I PH ft 6 cd OT o o d CD o cd OT o X I oa. PH I o c cd >^  o o CJ F I G U R E 3.4: Summary of the reactivation profiles of BgaX. Values of V£eact have been scaled relative to the maximum. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 58 J I L in o cd a o t- o 1 CD I xi PH CO 1 CD 1 CD Pi T 3 G T3 6 OS 1 oa. i 6 so cd OT o cd cj   o M o o glu 'ao 1 H-> CJ 'sb 1 glu <o. i cd oa. 1 Cu CO 1 Id o &o 1 < & 1—1 PH 1 -—' o OH 1 <c cd cj I cj < CD KI o o >, X! O 01 I CJ C\2 CD o CD T) m O £ 2 O i 1 CD CJ I 8 CD s CD CD ' O T T J o OT o o >•> i X i <30. PH xi 1 PH o < 1 1 CH CD 1 1 cd CJ OT o f—H H-> M CJ O cd c cd Id ao o 1 1 .—i PH 1 1 CH CD Id ' M CJ o £ 2 <afl O 1 Q CD CJ PH Z Q o SH + J C o CJ cd O F I G U R E 3.5: Summary of the reactivation profiles of BgaC. Values oiV„eact have been scaled relative to the maximum. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 59 CD CD CD X i X i OT OT OT O o o CJ -Q -a O o ,—( CD 1 CD o <=a. o 1 Qa.  02. PH PH CD 1 1 X PH I OH G CO PH 2 ; CD X i 'OT o O 1 Gn & CD O CD CD X i 'w o >^  X I 03. PH 2 ; CD X i • i—i w o O G 'ab PH o <C I PH CD XI 'm o -3 o X I 00-PH Q I m_ CD OT O o G "3D >> x o CD XJ I CD XJ ' O T o X I 02. CD CD X ! 'w O X I Q PH SH I a o H-> G o cj F I G U R E 3.6: Summary of the reactivation profiles of Man2A. Values of V£eact have been scaled relative to the maximum. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 60 CD CD CD CD T ) T J T ) m m m O o o o o • rH CJ qo qo ; — | lab 'ob CD Cu CD 1 CJ O C Q . 1 1 C Q . 1 C Q . Ph i 11 Cu Cu 2 Q 1 OH CD W O J O o -l-> c CD b O 0) T ) • t—i K> o X I CQ. Cu I OH CD CD CD KI T J T l O cj ' K I w O O * J -t-> "bb o C J CO CO cd Cu bo Cu | PH I PH o - 1 - " C O CJ F I G U R E 3 . 7 : Summary of the reactivation profiles of Abg. Values of y o r e a c t have been scaled relative to the maximum. It was necessary to use a logarithmic scale in order to adequately compare y o r e a c t values of the acceptors. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 61 CD CD CD CD CD "2 w T3 K> sid qp sid tos o u H-> o O o 1 o o C 03. 1 luc 1 cd c o o o X I CJ luc 1 CJ d^ c o 1—1 1 bO bO CU CD 03. 3 i 1 )-G >^  o 1 o OH 03. )-G OX a 0Q_ | i Ph CO 1 de CD CD DH I T 03. o T 03. Id o F I G U R E 3.8: Summary of the reactivation profiles of CelB. Values of yoreac* have been scaled relative to the maximum. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 62 CD X I •r—l w O o I—I CD CJ I Q CD S CD CD X J X J • 1—1 OT OT O O >> X 1 X 1 1 oa_ i oa. PH PH 2 1 2 t 1 DH i o CD .tol CD CD CD X ) .tol X J OS X J OT c OT H-> OT o o O o O o CO o o o CD CD CD CJ 1 O I O 1 I <tL l oa. 1 oa. PH CD PH 2 S 2 Q 1 DH cO o F I G U R E 3.9: Summary of the reactivation profiles of Cex. Values of Vgeact have been scaled relative to the maximum. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 63 o o o CO o CD 5^  o Xi cu 01 C Q . I CJ o I CO I C Q . 13 o cu OT o <! o 3 I C Q . Cu 2 CD CO CD OT w c O o - * J * J '3 o CJ c (0 d 1—f OT (0 O bO +-> CJ cd cd bO O < i i 2 <D OT o 'I—I £ 2 cd cd CD ' O T o o I C Q . CU 2 Q o S-c o cj F I G U R E 3.10: Summary of the reactivation profiles have been scaled relative to the maximum. of HBG. Values of V£eact C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 64 For each glycosidase, excluding HBG, the l / r e a c * values associated with the dis-accharide acceptors (and especially those derivatized with an aryl group) generally ranked highest among each group of positive hits. This was anticipated, as the addi-tional sugar moiety is expected to provide further favorable contacts with the aglycone site of each enzyme. The glycosidic linkage between sugar units in the disaccharide ac-ceptors did not always significantly influence the degree of productive binding between acceptor and aglycone site. For the glycosidases CelB and BgaX, values of l / r e a c t asso-ciated with enzyme reactivation are similar for the acceptors Gal-/?-(l-4)-Glc-/3-SPh and Gai-/3-(l-3)-Glc-/3-SPh. A dependence was seen however on the internal acceptor linkage with the reactivation of BgaC and HBG. These enzymes showed an initial positive hit for the /3-(l-3) linked Gal-Glc thiophenyl glycoside, yet reactivation was less than that of the control for the /3-(l-4) isomer in the large scale screen (Table 3.1). A more substantial effect on the reactivation of BgaC was seen in the presence of maltotriose whose value for l / r e a c t was ca. 34-fold greater than that of the control, and ca. 4-fold greater than any other acceptor. This particular result is interesting in that it illustrates the need to screen a large number of compounds when determining the most appropriate transglycosylation acceptors for a given glycosidase. It was not anticipated that there would be substantial binding between the a-(1-4) linked glucose units of maltotriose and the aglycone site of an enzyme optimized for the hydrolysis of /3-(l-3) linked terminal galactose residues. The best acceptors for the remaining glycosidases studied were much less surpris-ing: all are derivatives of cellobiose, or for the case of BgaX, the (3-(l-3) and /3-(l-4) linked Gal-Glc thiophenyl glycoside isomers. The values of V0react associated with D-glucal and pNPCell for the cellulase Cex C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 65 are slightly lower than that for the control. These findings are believed to be the result of the aforementioned problem of the acceptors also acting as substrates for the glycosidase. Over the course of reactivation, pNPCell was likely hydrolyzed, and hence converted to less effective acceptor. Upon assaying for enzyme reactivation, the presence of cellobiose likely competed for the active site of Cex, resulting in an apparent lower activity. The acceptor D-glucal likely behaved in a similar manner. Cex has been shown to be capable of hydrolyzing aryl glucosides [53], and is therefore likely to be able to use D-glucal as a substrate, based on the finding that Abg, a glucosidase, is able to hydrate this sugar to form 2-deoxyglucose [54]. However, as was mentioned earlier for those acceptors whose reactivation profiles deviated from a first order expression after a period of time, pNPCell and D-glucal were still identified as good acceptors. 3.2.3 Positively screened glycosides as transglycosylation acceptors The true test of the utility of the screen for transglycosylation acceptors comes from being able to perform transglycosylation reactions with the postitively screened glycosides. Four of the enzymes studied (BgaX, BgaC, Abg, and Man2A) were used in preparative scale reactions with a pNP-glycoside specific for their glycone site as the donor, and one of the positively screened glycosides acting as the acceptor. The isolated products from each reaction were acetylated, isolated, characterized by NMR, and compared to transglycosylation products from the literature (Table 3.3). Results of all but one of the experiments listed in Table 3.3 agree with the results of the screening procedure. That is, most of these sugars were identified as being capable of acting as acceptors in transglycosylation reactions. One exception does exist however, C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 66 T A B L E 3.3: Tranglycosylation reaction products from this study and the litera-ture. E N Z Y M E [ R E F ] D O N O R " A C C E P T O R " P R O D U C T S (% Y I E L D ) 0.(1-3) Hl-4) linked linked Abg pNPGal gentiobiose 21.4% Abg glycosynthase [26] a-Gal-F pNPGlu 84% Abg glycosynthase [26] a-Gal-F pNPXyl 81% Abg glycosynthase [26] a-Gal-F pNPMan 66% Abg glycosynthase [26] a-Gal-F pNPCell 92% Man2A pNPMan PhGlc 11.8% Man2A [28] pNPMan pNPMan 3.5% 6.7% Man2A glycosynthase [28] a-Man-F pNPMan 1.4% 6.5% Man2A glycosynthase [28] a-Man-F pNPGentiobiose » 80%" Man2A glycosynthase [28] a-Man-F pNPCell « 30%6 BgaC pNPGal maltotriose 30.0% BgaC [55] pNPGal GlcNAc 12.2% BgaC [55] pNPGal GalNAc 10.1% BgaX pNPGal pNPXyl 6.1% BgaX [45] pNPGal GlcNAc 22.4% BgaX [45] pNPGal GalNAc 17.3% 7.41% "All glycosides, excluding maltotriose (a-D-Glu-(l-4)-a-D-Glu-(l-4)-D-Glu) are /?-D- configured unless noted otherwise. 6Yield was estimated from T L C analysis and represents all products formed. which is discussed below. It is difficult to draw direct correlations between the initial rate of enzyme reac-tivation in the presence of a particular compound and the product yield of a trans-glycosylation reaction in which that same compound is used as an acceptor. The yield of such a reaction is dependent on other factors including the concentration of donor and acceptor used, the time period over which the reaction is performed, and, if a glycosidase is used, the susceptibility of the products to hydrolysis. However, the results of Table 3.3 support the basic notion on which the screening procedure is founded: that is, adequate acceptors for a given glycosidase or glycosynthase can be identified by comparison of the rate of reactivation of an inhibited glycosidase in the presence of the compound in question. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 67 Note that analysis of enzyme reactivation in the presence of an acceptor does not give any indication as to the number of discrete products or the glycosidic linkages contained therein that are formed in a corresponding transglycosylation reaction. This must be deduced by actually performing the desired reaction. It was discussed in the previous section that one drawback to the screening strategy is the inability to screen some glycosides that are substrates for the enzyme under study. This shortcoming is illustrated in Table 3.3 by the ability of pNPMan to act as an acceptor for Abg glycosynthase when this glycoside was tested as a negative hit. Wild type Abg is capable of using pNPMan as a substrate {kcat/Km=Q s~lmM~l [10]), which suggests that this sugar was most likely hydrolyzed over the course of the reactivation study to result in a negative hit. The data of Table 3.3 indicates that pNPXyl, GlcNAc, and GalNac are all ca-pable of acting as acceptors for BgaX. The first two of these sugars were identified as positive hits in the screening procedure; however, GalNAc was observed to be a negative hit. It is unclear as to why GalNAc would register as negative; since it is not derivatized with a pNP group, it should not be degraded over the course of the experiment through enzyme-catalyzed hydrolysis. It is possible that the transglycosy-lation product formed from enzyme reactivation by GalNAc inhibited active enzyme during the experiment, causing a false negative result. Clearly, the combined yield of 24.7 % of the transglycosylation reaction in the presence of GalNAc indicates that this glycoside is capable of acting as an adequate acceptor. This false negative result was the only such observed for the screening process, excluding the aforementioned possibility of enzyme substrates. C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 68 3.3 Conclusions Although the detection of a false negative result is detrimental to the integrity of the developed methodology, it does not compromise the original intention, to screen a large number of compounds to identify those that will act as acceptors in trans-glycosylation reactions. Such identification has been readily demonstrated by the ability of the several chosen positively screened glycosides to act as acceptors in the transglycosylation reactions performed, and by the reactions documented from the literature. In general, aryl glycosides were shown to act as better acceptors as compared to their parent carbohydrates. Some specificity was shown regarding the substitution of the aryl moiety of such glycosides and appears to be dependent on the particular enzyme under study. Similarly, disaccharides were shown to generally act more fa-vorably as acceptors compared with monosaccharides, a consequence believed to be the result of additional favourable interactions with the enzyme's aglycone site. Note however, that the sensitivity towards the linkage type of disaccharides with respect to aglycone specificity varies between enzymes. The described screening procedure has one drawback in that it is not a reliable approach to evaluate the potential of a substrate for a given glycosidase to also act as a transglycosylation acceptor. Considering that there are a vast number of compounds (not necessarily glycosides) that one may wish to use as acceptors, this setback, while detrimental, does not detract from the overall utility of the methodology. The generally low number of positive hits resulting from all potential acceptors examined (16-36%) illustrates the necessity of screening a large number of compounds to identify the maximal number of acceptors for a given glycosidase. The developed screening strategy is optimal for such a task; it is easily performed in a reasonable C H A P T E R 3 Screen of Potential Transglycosylation Acceptors 69 amount of time, and requires the use of few specialized reagents and equipment. The synthesis of fluorinated glycosides in this laboratory and others has provided a foun-dation for the development of mechanism based inactivators for numerous retaining glycosidases which could be used in the screening process. Although the methodology described in this study was used to screen for potential transglycosylation acceptors, it also provides a strategy by which to generally probe the specificity of the aglycone site of a retaining glycosidase. This information would provide insight into architechture of an enzyme's aglycone site, which could be useful in future studies such as those that correlate structure and function. C H A P T E R 4 Materials and Methods 4.1 The characterization of BgaX 4.1.1 General All buffers, substrates, and reagents were purchased from either Sigma-Aldrich Co. or Aldrich Chemical Company unless noted otherwise. Galacto-imidazole was a gift from A. Vasella (Institut fur Organische Chemie, ETH-Ziirich). pNPGalNAc was a gift from S.J. Williams (Department of Chemistry, UBC). Enzyme activity assays were done with a Unicam UV4 ultraviolet/visible spec-trometer; the spectrometer and cuvettes'were pre-equilibrated to 37°C by the use of a circulating water bath. Polystyrol and acryl 1 mL disposable cuvettes were used for all spectroscopic measurements, excluding the measurement of activity of BgaX with pNP-a-L-Ara, pNPFuc, pNPGalNAc, pNPGlc, and pNPXyl; for these experiments, 200 fiL quartz cuvettes were used. Protein sequence alignments were done with the NCBI BLAST server site [56, 57]. N-terminal protein sequencing was performed by the Nucleic Acid-Protein Service Unit, UBC. Curve fitting was done with Grafit [58]. 70 C H A P T E R 4 Materials and Methods 71 4.1.2 The molar absorptivity of 2,4-dinitrophenol and p-nitrophenol A sample of pNP was dried in vacuo and used to make a stock solution in water (3.31 mM), aliquots of which were added via syringe to 800 //L of the appropriate 50 mM buffer to final pNP concentrations ranging from 41.0-80.7 pM (pH 4.0-6.5 = citrate buffer; pH 6.5-8.0 = sodium phosphate buffer; pH 8.0-9.0 = glycine buffer) . The absorbance at 400 nm of each sample was measured and used to calculate the molar absorptivity (e) from Beer's Law: A = edc (4.1) where A = absorbance at 400 nm e = molar absorptivity (cm - 1 m M - 1 ) d = the cell pathlength (1 cm) c = the concentration of pNP (mM) The resultant values of e were fit to the following equation [58]: IA - Ul ( I ( ) ( P " - / < ' ^ ) ) where LI and L2 = e for the fully protonated and deprotonated forms of pNP respectively The pK a of pNP and e at pH 6.0 was extrapolated from the curve fit to the above equation. The determination of e of DNP at pH values of 3.5-6.5 was performed in the same manner as that described for pNP. Values of A 4 0o at DNP concentrations of 20.0-90.0 fM in 150 mM NaCl, 50 mM buffer (as above) were used to construct the e C H A P T E R 4 Materials and Methods 72 versus pH plot. The curve fit to Equation 4.2 was used to determine the pK a of DNP and e values at each pH. The calculated values of £ for pNP and DNP (Table 4.1) were used in successive studies to convert Vmax from units of AA 4 0 o/min to mM substrate hydrolyzed/min. T A B L E 4.1: Values of e for pNP and DNP p H 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 e of p N P (cm 1 m M - X) 2.30 2.37 2.611 3.32 5.21 9.05 13.84 17.17 e of D N P ( c m - 1 m M " X) 13.67 15.58 16.29 16.52 16.60 16.63 16.64 16.64 4.1.3 Source, expression and purification of BgaX M o d i f i c a t i o n o f gene All gene manipulations were done by Janine Foisey (Department of Microbiology, UBC). The gene encoding BgaX without its amino-terminal 21 amino acid signal se-quence coding region (pCT/3-gal-100) was obtained as a construct in the New England Biolabs in-house vector pAGR3 from Dr. Christopher Taron (Department of Biochem-istry, University of Illinois at Urbana-Champaign). A new pCT/3-gal-lOO construct that incorporated a 6-histidine tag at the carboxy-terminal of the protein was engi-neered in the vector pTuglON18 through the polymerase chain reaction (PCR) using the primers of Figure 4.1. The BgaX gene construct was transformed into E. coli JM101 via electroporation. C H A P T E R 4 Materials and Methods 73 5' GTC GAA AAC AGC GGC CGG ATC AAT 3' 3' GTG GGG TCG CAC GCG CCG GTA GTG GTA GTG GTA GTG ATT CGA TCG TTC GAA TCT GAT 5' F I G U R E 4.1: The primers used in PCR to fuse a 6-His tag to BgaX. Expression Expression of BgaX was done by L. Gal (Department of Microbiology, UBC). E. coli cells containing the BgaX construct were plated onto LB/Amp media (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 100 izg/mL ampicillin), and a single resultant colony used to inoculate a 30 mL Typ/Amp starter culture (16 g/L tryptone, 16 g/L yeast extract, 5 g/L NaCl, 100 fig ampicillin, pH 7.0). After incubation for 5.5 hr at 37°C, the starter culture was used to inoculate a 2 L Typ/Amp culture. After incubation at 30°C for 3 hr, isopropyl-/3-D-thiogalactoside was added to a final concentration of 0.1 mM, and the culture was incubated at 30°C until OD600 = 0.5. The cells were harvested and spun at 4°C for 15 min at 44,000 x g to produce the cell paste. P u r i f i c a t i o n The cell paste was suspended in 50 mM sodium phosphate buffer, 0.5 M NaCl, pH 6.0 (start buffer) (3.8 mL/g). Phenylmethylsulfonyl fluoride (final concentra-tion = 0.25 mM) and /3-mercaptoethanol (final concentration = 6.7 mM) were added and the suspension was French pressed 3x using an SLM Aminco French Pres-sure Cell Press in a 20-K cell. The lysed cells were centrifuged at 44,000 x g for 1 hr, and the supernatant decanted and filtered through a 45 fim membrane (final vol-ume ca. 45 mL). The clarified supernatant was loaded onto a Pharmacia Biotech X16 column packed with Pharmacia Biotech Chelating Sepharose Fast Flow gel (18 mL) C H A P T E R 4 Materials and Methods 74 charged with N i 2 + according to the manufacturer's instructions, and pre-equilibrated with start buffer. BgaX was eluted over 10 column volumes with a linear imidazole gradient of 0-100 mM. Fractions that showed /3-galactosidase activity (determined as described below) were pooled (VT = 130 mL) and dialyzed against 2 x 2.5 L of 20 mM sodium phosphate buffer, 50 mM NaCl, pH 6.0 (SP buffer) then concentrated in 30,000 molecular weight cut-off Amicon Centriprep concentrators and loaded onto two pre-packed 5 mL Pharmacia Biotech Hi-Trap SP columns connected in tandem, and pre-equilibrated with SP buffer. BgaX was eluted with a linear NaCl gradient of 50-350 mM over 15 column volumes. The purified BgaX appeared as a single band in silver-stained SDS-PAGE. N-terminal sequencing of the wild type BgaX (a gift from J. Foisey, Department of Microbiology, UBC) and the purified 6-His tagged BgaX proteins (ATPESW) were consistent with the SWISS-PROT entry (P48982) [59] for this enzyme . 4.1.4 pH Stability Very preliminary preparations of BgaX were kept in 50 mM sodium phosphate, pH 7.0. Enzyme activity remained constant over at least 5 hr at room temperature at this pH. The activity of BgaX at pH 7.0 was therefore used as a reference by which to determine the stability of this enzyme over a range of pH values. Buffers used for this study were 150 mM NaCl in 50 mM citrate (pH 4-6) and sodium phosphate (pH 6-8). BgaX (0.12 mg) was added to each pH solution (Vr= 200 fjL), incubated at room temperature for 2 minutes, and then assayed for activity as described below. The residual activity as compared to activity at pH 7.0 was plotted against pH. C H A P T E R 4 Materials and Methods 75 4.1.5 p H Dependence The parameters kcat and K m for the hydrolysis of DNPGal by BgaX were deter-mined as described below (Hydrolysis kinetics) at pH values ranging from 4.5-8.0. The initial velocity of hydrolysis was determined after the addition of BgaX (0.94 mg) to aliquots of DNPGal at five different concentrations (0.016-1.39 mM) in 0.1% BSA (w/v) (total volume = 810 /iL). Buffers used at each pH value (pH 4.5-6.0 = citrate buffer, pH 6.0-8.0 = sodium phosphate buffer) were 50 mM and contained 150 mM NaCl. The initial velocity of each reaction for a given pH value was plotted against substrate concentration and fit to the classical Michaelis-Menten equation to derive Vmax and Km, and ultimately, kcat. The kinetic parameters kcat and kcat/Km were then plotted against pH and fit to y = 1 D (2 .pff -pKi , r ip^+io (pH-pK n i ) + 1 where y = kCat or kcat/Km and L is the upper limit. 4.1.6 Kinetics Measurement of enzyme activity The rate of hydrolysis of nitrophenyl-glycoside substrates were determined by the continuous spectrometric monitoring of the release of pNP or DNP at 400 nm (AA4oo/min) over 1-2 minutes. Spectrometer cuvettes contained 0.1% BSA (w/v) in 50 mM sodium phosphate buffer, pH 6.0 (buffer A) unless noted otherwise. Assays were initiated by the addition of a 5 fiL enzyme aliquot. Unless stated otherwise, the activity of BgaX was determined with 1.2 mM pNPGal at 37°C. C H A P T E R 4 Materials and Methods 76 Hydrolysis kinetics The activity of BgaX was determined as described above at at least five different concentrations of 0.007-3.00 mM pNPGal, 0.01-0.52 mM DNPGal, 0.3-2.2 mM pNP-a-L-Ara, 0.5-12.6 mM pNPFuc, 0.4-3.4 mM pNPGalNAc, 1.5-49.7 mM pNPGlc, and 1.8-15.0 mM pNPXyl. The resultant measured velocities (AA^o/min) were plot-ted against substrate concentration and fit to standard Michaelis-Menten kinetics (v = ^""[gj) to determine Km and Vmax, and ultimately, kcat. Inactivation by 2FDNPGal 2FDNPGal (1.15-46.0 fiM) in BSA-free buffer A (final volume = 100 fiL ) was incubated in a 20°C water bath for 5 minutes. BgaX (9.7 fig) was added to initiate inactivation. Aliquots of 5 fiL of this inactivator mix were removed over time and residual enzyme activity was assayed as described. The fractional loss in activity was plotted against time and fit to a first order exponential decay The observed rate constants (k°bsact) so derived were replotted in double reciprocal form with inhibitor concentration and fit to k°„act = k£°+$ to determine the inactivation rate constant (kinact) and inhibitor dissociation constant (Ki) of 2FDNPGal with Protection from inactivation The Ki of galacto-imidazole with BgaX was determined by the measurement of the initial velocity of hydrolysis, as described above, by the addition of BgaX (9.7 fig) to five concentrations of pNPGal (7.0-91.0 fiM) in the presence and absence of 7.2 nM galacto-imidazole (P) (volume = 805 fiL). The measured initial velocities and sub-strate concentrations were plotted in Lineweaver-Burk form and and the slopes fit to BgaX. C H A P T E R 4 Materials and Methods 77 ~ siopeXiope0-\ t o derive Ki (where slope0 = slope of the Lineweaver-Burk plot in the absence of P, and slope; = slope of the Lineweaver-Burk plot in the presence of P)-Protection of BgaX from inactivation by 2FDNPGal was shown by the determi-nation of k°bsact (determined as described above) of BgaX (9.7 //g) with 46 /J.M of 2FDNPGal (in 100 //L BSA-free buffer A) in the presence (Jfcgs) and absence (kf") of 40 nM galacto-imidazole and comparison of k°bs/k°bs to the value predicted by the following equation: kJl= K ' + l'1 ( 4 3 ) K , ( 1 + M ) + [ „ ( 4 3 ) C a t a l y t i c c o m p e t e n c e o f i n a c t i v a t e d species : Generation of the inactivated enzyme or 'I-mix': BgaX (2.42 mg) was incubated with 2FDNPGal (207 mmol) in BSA-free buffer A (volume = lmL ) until < 5% of the original activity remained. The inactivated enzyme was concentrated using a 30,000 molecular weight cut-off Millipore Ultrafree 0.5 centrifugal filter and tube assembly to a volume of 10-20 fiL, and fresh BSA-free buffer A was added to a final volume of 200 //L. This process of concentration and dilution was repeated until all excess inactivator was removed, as determined by the maintenance of full activity of an aliquot (5 fjL, 0.01 mg) of non-inactivated enzyme sample diluted one half with the inactivated species or T-mix'. The concentration of inactivated enzyme in the I-mix was determined by absorption at 280 nm (e= 1.97 mL m g - 1 cm - 1 ) 1 , and used to calculate the maximal activity possible (V0) if the enzyme were completely active. lAs determined by amino acid sequence: e = ( (# Trp)(5,500) + (# Tyr)(1,490) + (# C y s ) ( 1 2 5 ) ) / M W [60] C H A P T E R 4 Materials and Methods 78 Determination of catalytic competence: A dilution of the BgaX I-mix (24 yug in 50 /xL) was incubated at 20°C in buffer A and aliquots were removed over time and assayed as described. The resultant gain in fractional activity was fit to a first order rate equation [y- = A{\ — e~kreactt) + C^j and the rate constant for turnover of the glycosyl-enzyme intermediate (kreact) determined. 4.1.7 Identification of the catalytic nucleophile of BgaX D i g e s t i o n o f i n a c t i v a t e d e n z y m e : BgaX (14.5 ng) was incubated with 2FDNPGal (25.8 mmol) in BSA-free buffer A (volume = 65 pL) until < 5% of the original activity remained. An aliquot (10 zzL) of the inactivated enzyme was frozen and kept at -20°C until analyzed by mass spectrometry. The inactivated species (50 fiL) was added to pepsin in a ratio of ca. 12:1 (enzyme:pepsin) in 200 mM sodium phosphate, pH 2.0 (volume = 78 fjL). The digestion was done at room temperature for 2 hrs; the sample was then frozen and kept at —20°C until analysed by mass spectrometry. E l e c t r o s p r a y mass s p e c t r o m e t r y All mass spectrometry was done by S. He (Department of Chemistry, UBC). Mass spectra were recorded on a Perkin-Elmer Sciex API 300 triple quadrupole mass spec-trometer with an ion spray ion source. The spectrometer was interfaced with a Michrom BioResources Inc. Ultrafast Microprotein Analyzer which separated pep-tides via reverse phase HPLC. Digested proteins were loaded onto a Reliasil C18 column (1 x 150 mm) equilibrated with solvent A (0.05% trifluoroacetic acid (TFA), 2% acetonitrile in water). Peptides were eluted over a 0-60% gradient of solvent B C H A P T E R 4 Materials and Methods 79 over 60 minutes, followed by 100% solvent B over 2 minutes (solvent B: 0.045% TFA, 80% acetonitrile in water). The pump rate was 50 //L/min. Spectra were obtained in the single quadrupole scan mode (LC/MS) or the tandem MS daughter ion scan mode (MS/MS). In LC/MS, the quadrupole mass analyser was scanned over a mass to charge ratio (m/z) of 300-2400 Da with a step size of 0.5 Da and a dwell time of 1 ms per step. The ion source voltage (ISV) was 5 kV and the orifice energy (OR) was 50 V. In MS/MS, the spectra were obtained by selectively introducing one of the parent peptides of m/z= 869.0 and 951.0 from the first quadrupole (Ql) into the collision cell (Q2) and observing the daughter ions in the third quadrupole (Q3). The scan range of Q3 was 50-1920 Da; step size, dwell time, and OR as above; ISV was 4.8 kV. 4.1.8 The mutant H6-E260A BgaX E. coli cell paste containing H6-E260A BgaX was a gift from J. Foisey (Department of Microbiology, UBC). The mutant protein was purified by a Ni2+-chelating column as described for H6-BgaX. The protein was «90% pure as estimated by silver stained SDS-PAGE. 4.1.9 Measurement of activity of H6-E260A BgaX The activity of H6-E260A BgaX was determined with and without azide present. Spectrometer cuvettes contained 790 //L 50 mM 2-(./V-morpholino)ethane sulfonic acid (MES), pH 5.0, 0.1% BSA (w/v), 1.0 mM DNPGal, and either 0 or 2 M azide. The background hydrolysis of DNPGal was determined over 1-2 minutes, then a 5 jih aliquot of the mutant enzyme was added and the initial velocity of the reaction determined. C H A P T E R 4 Materials and Methods 80 4.2 Large-scale screening of potential acceptors 4.2.1 General All enzymes, excluding BgaX, were expressed and purified by other laboratory members or collaborators (Table 4.2). The activity of each enzyme was determined as described for BgaX (§4.1, Measurement of activity) in the buffer system and with the substrates and concentrations of Table 4.2 (in 775-900 /JL). Enzyme inactivators were gifts from members of this laboratory: 2FDNPGal, 2FDNPGlc, and 2FManF (L. Mackenzie), 2FDNPCell (D. Zechel), 2FDNPGlcA (A. Wong). Potential acceptors were purchased from Sigma-Aldrich Co. or Aldrich Chemical Company or were gifts TABLE 4.2: Conditions for the measurement of activity of the enzymes screened. E N Z Y M E ( S O U R C E ) B U F F E R S U B S T R A T E [ P U R I F I C A T I O N P R O T O C O L ] BgaX 50 mM NaPi a , pH 6.0 1.2 mM pNPGal BgaC (L. Gal t 6) 50 mM NaPi, pH 7.0 0.94 mM pNPGal Abg (K. Rupitz* [21]) 50 mM NaPi, pH 7.0 0.50 mM pNPGlc Man2A (D. Stall* [28]) 50 mM NaPi, pH 7.0 0.35 mM pNPMan Cex (K. Rupitz* [61]) 50 mM NaPi, pH 7.0 1.2 mM pNPCell CelB (C. Dupont5 [62]) 50 mM NaPi, pH 6.5 0.50 mM DNPGal HBG (J.H. Grubb^ 100 mM NaAc c, pH 4.5 1.83 mM pNPGlcA and W . S . Sly11 [63, 64]) "Sodium phosphate. 6 L. Gal incorporated a 6-His tag to the carboxy terminal of BgaC and purified the enzyme in an unpublished protocol that was very similar to that reported here for BgaX. cSodium acetate. * Department of Microbiology, UBC. ^Department of Chemistry, UBC. §INRS Institut Armand-Frappier, Centre de recherche en microbiologie et biotechnologie ^E.A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine C H A P T E R 4 Materials and Methods 81 from members of this laboratory: DNPFuc (J. McCarter), pAcPh /3-Glc (Q. Wang) C0 2 Me P-Glc (Q. Wang), D-glucal (L. Mackenzie), 1-cyano-D-glucal (E. Lai), 1,2-dicyano-D-glucal (T. Harvey), DNPCell (H. Prade), Me a-Cell (V. Ferro), Me /?-Cell (V. Ferro), Gal-/3-(l-3)-Glc-£-SPh (H. Prade), Gal-0-(l-4)-Glc-0-SPh (H. Prade), 2,5-DNPX 2 (D. Zechel). Stock solutions of potential acceptors were made in water to a final concentration of ca. 50 mM; some of these solutions were saturated. Thin layer chromatography (TLC) was done with aluminum backed silica 60 F254 from E.M. Science. All TLC's were examined under UV light and then stained with 10% H 2 S O 4 in MeOH and charred. Chromatography was done with 230-400 mesh silica in ca. 15 cm columns. NMR spectra were recorded in CDC13 on a Bruker AC 200 or Bruker WH 400 and are referenced to the solvent peak. Where required, interpretations were supported by COSY experiments. 4.2.2 Screening A: large scale screening I-mixes of each enzyme were prepared as described for BgaX (§4.1.6, Catalytic competence of inactivated species) with the enzyme and inactivator amounts listed in Table 4.3 (volume = 0.5-1.0 mL; buffer system as in Table 4.2). An aliquot of each I-mix (Table 4.3) plus buffer and each compound to be screened (Tables 3.1 and 3.2 ) were added to the wells of a 96-well plate so the final concentration of the potential acceptors was 30 mM or 60% saturation (final volume = 50-100 jih). Controls of (i) non-inactivated enzyme and (ii) inactivated enzyme, each in buffer alone, were included. The plates were incubated at room temperature for the times indicated (Table 4.3). The amount of enzyme reactivated after incubation was determined by the measurement of enzyme activity: 100 //L of the corresponding assay substrate C H A P T E R 4 Materials and Methods 82 T A B L E 4.3: Conditions for the screen of potential acceptors. E N Z Y M E F O R I N H I B I T O R E N Z Y M E I N I N C U B A T I O N A S S A Y I N A C T I V A T I O N E A C H W E L L T I M E S U B S T R A T E 0 340 ng BgaX 258 mmol 2FDNPGal 1.60 Mg 19 hr 2.5 mM pNPGal 440 Mg BgaC 155 mmol 2FDNPGal 5.67 Mg 17 hr 2.7 mM pNPGal 470 fig Abg 122 mmol 2FDNPGlc 1.73 Mg 18 hr 2.0 mM pNPGlc 80 ng Man2A 187 mmol 2FManF 0.26 Mg 2.5 hr 0.35 mM pNPMan 310 Mg Cex 189 mmol 2FDNPCell 4.55 Mg 16.5 hr 2.3 mM pNPCell 60 /xg CelB 60 mmol 2FDNPCell 0.75 Mg 3.5 hr 0.75 mM DNPCell 121 Mg HBG 100 mmol 2FGlcAF 1.39 Mg 180 hr 2.05 mM pNPGlcA "Final concentration in each well upon assaying for activity. (Table 4.3) was added to each well to the final concentrations indicated, and the release of the pNP or DNP phenolate ion was measured with a 96-well plate reader either by: (i) taking single absorbance measurements of each well at 400 nm over 0.5-6 hr (using a Bio-Rad Model 2550 EIA Reader), or (ii) measuring the continuous change in absorbance at 400 nm of each well at 400 nm over 10-20 minutes (using a Molecular Devices Spectra Max 190 Reader), the latter procedure being preferred. Tested compounds were classified as a positive hit if they induced greater enzyme reactivation as compared to the control of reactivating enzyme in buffer alone. 4.2.3 Screening B : analylsis of enzyme reactivation BgaX, BgaC, Man2A, Abg, Cex, CelB, and HBG I-mixes were prepared as de-scribed in §4.1.6 (Catalytic competence of inactivated species) with the conditions described in Table 4.4. I-mixes were added to buffer and those compounds identified as positive hits (from the large scale screening described above) in eppendorf tubes so the final concentration of each tested compound was 30 mM or 60% saturation C H A P T E R 4 Materials and Methods 83 (volume= 50-100 //L). All such mixtures were incubated in a water bath at 37°C, excluding BgaX (incubation at 20°C). Controls of non-inactivated enzyme were in-cluded to account for enzyme death over time. Aliquots of 5 /LtL of each sample were removed over time and assayed for activity as described above for BgaX (§4.1.6 Mea-surement of activity) with the buffers and substrates of Table 4.2. The fractional gain in activity was fit to a first order rate equation and values of k$*ns and A were extrapolated and used to calculate the initial rate of reactivation (yjeact = Ak°^ns). For BgaC and Cex, reactivation was so slow that over the course of the experiment, the data collected was contained within the linear portion of the first order reactivation expression; therefore, values of Vorea0t for these enzymes were obtained from a direct linear fit of the data. T A B L E 4.4: Conditions for the analysis of the effect of each postive hit on enzyme reactivation^ E N Z Y M E F O R I N H I B I T O R E N Z Y M E I N I N A C T I V A T I O N E P P E N D O R F 2.4 mg BgaX 207 mmol 2FDNPGal 50 ug 5.5 mg BgaC 103 mmol 2FDNPGal 175 ug 1.8 mg Abg 612 mmol 2FDNPGlc 40 ug 0.1 mg Man2A 312 mmol 2FManF 4 ug 0.6 mg Cex 94 mmol 2FDNPCell 140 ug 0.3 mg CelB 40 mmol 2FDNPCell 2/xg 2.8 mg HBG 98 mmol 2FGalAF 140/xg C H A P T E R 4 Materials and Methods 84 4.2.4 Preparative scale transglycosylation reactions Enzyme in buffer (Table 4.5) was incubated with acceptor (A) and a portion of donor (D) (D:A « 1:5) at room temperature so the final concentration of A was 40-50 mM (volume = 5-8 mL). Reactions were monitored by TLC (solvent: 7:2:l=EtOAc:MeOH:H20).. The rest of D was successively added in ca. 1/4 amounts after approximately 90% of the D in the reaction mixture was depleted (estimated by TLC). After total depletion of D (4-14 hr), reactions were lyophilized and acetylated. Acetylation of transglycosylation products Transglycosylation products were acetylated with either (i) acetic anhydride (Ac 20) or (ii) l-1 3C-acetyl chloride (l- 1 3C-AcCl) according to the following methods: (i) A C 2 O (3 mL) was added under N 2 to the mixture of transglycosylation products in dry pyridine (6 mL) at room temperature. Once the reaction was complete by TLC (solvent: l:l=EtOAc:hexanes), the solvent was removed by the addition of toluene and evaporation in vacuo. (ii) l - 1 3 C-AcCl (150-200 fiL) was added under N 2 to the mixture of transglyco-sylation products in dry pyridine (2 mL) at room temperature. Once the reaction T A B L E 4.5: Conditions for preparative scale transglycosylation reactions. E N Z Y M E D O N O R " A C C E P T O R B U F F E R S Y S T E M BgaX (0.194 mg) 73.2 mg 54.0 mg 50 mM NaPi, pH 6.0 BgaC (0.405 mg) 88.1 mg 220.6 mg 50 mM NaPi, pH 7.0 Abg (0.540 mg) 77.3 mg 71.5 mg 50 mM NaPi, pH 7.0 Man2A (0.160 mg) 48.7 mg 66.7 mg 50 mM NaPi, pH 7.0 "Total amount of donor added over the course of the reaction C H A P T E R 4 Materials and Methods 85 was complete by TLC (solvent: 3:l=EtOAc:hexanes), the solvent was removed by the addition of toluene and evaporation in vacuo. Water and EtOAc were added to the residue (ca. 15 mL of each) and successively washed with 1 M HC1, IM NaHC0 3 and saturated NaCl. The organic layer was recovered, and the solvent removed in vacuo. S e p a r a t i o n o f t r a n s g l y c o s y l a t i o n p r o d u c t s Acetylated transglycosylation products were loaded onto a silica gel column for chromatography in the minimum amount of CH 2C1 2 and eluted with a solvent system of 1:1 or 2:1 petroleum ether:EtOAc, or a combination thereof. Fractions of each prod-uct were pooled and the solvent removed in vacuo. The isolated products were dried in vacuo for ca. 48 hr, and submitted for NMR. Product yields are based on isolated products and are referenced to the total amount of donor used in each reaction. N M R d a t a o f t r a n s g l y c o s y l a t i o n p r o d u c t s 2,3,4,6- Tetra- O-acetyl-B-D-galactopyranosyl- (1—>3 )-2,4,6-tri-O-acetyl-a-D-gluco-pyranosyl-(l-^A)-2,3,6-tri-0-acetyl-a-D-glucopyranosyl-(l-^A)-2,3,6-tri-0-acetyl-a-T>-glucopyranose; lR NMR (400 MHz, CDC13): 6 5.71 (d, 1 H, J 1 | 2 8.1 Hz, H-l), 5.48 (dd, 1 H, J 2 i 3 10.1 Hz, J 3 ] 4 9.0 Hz, H-3), 5.38 (dd, 1 H, J 3 , | 4 , 9.7 Hz, H-3'), 5.33 (d, 1 H, J3»/ i4„, 3.3 Hz, H-4'"), 5.28 (d, 1 H, Jv,^, 4.1 Hz, H-l"), 5.22 (d, 1 H, JVf7, 4.0 Hz, H-l'), 5.04 (dd, 1 H, J 2,« i 3«i 10.4 Hz, H-2'"), 4.97-4.89 (m, 3 H, H-2, H-4", H-3'"), 4.78 (dd, 1 H, J2»,3» 10.2 Hz, H-2"), 4.69 (dd, 1 H, J 2 , i 3 , 10.3 Hz, H-2'), 4.61 (d, 1 H, Jv„r, 8.0 Hz, H-l'"), 4.47-4.38 (m, 3 H, H-6a, H-4', H-6a'), 4.24-4.12 (m, 5 H, H-6b, H-6b', H-6b", H-6a"', H-6b"'), 4.09-3.99 (m, 3 H, H-4, H-5, H-3"), 3.95-3.81 (m, 4 H, H-4', H-5', H-6b", H-5'"), 2.22, 2.14, 2.13, 2.11, 2.07, 2.03, 2.02, 1.99, 1.98, 1.97, 1.96, 1.93 (-12 s, 42 H, Ac). C H A P T E R 4 Materials and Methods 86 Para-nitrophenyl 2,3,4,6-tetra-0-acetyl-/3-D-galactopyranosyl-(l —> 3)-2,4-di-0-acetyl-p-D-xylopyranoside; lE NMR (400 MHz, CDC13): S 8.20 (d, 2 H, Ja>b 9.3 Hz, pNP), 7.06 (d, 2 H, Jc4 9.0 Hz, pNP), 5.37 (dd, 1 H, JA,# 3.2 Hz, H-4'), 5.17 (dd, 1 H, J 2 , 3 8.1 Hz, H-2), 5.16 (dd, 1 H, J 2 , > 3 , 10.5 Hz, H-2'), 4.99 (dd, 1 H, J3,A, 3.3 Hz, H-3'), 4.96 (ddd, 1 H, J 4 , 5 e ( ? 4.3 Hz, H-4), 4.56 (d, 1 H, Jv>2, 7.8 Hz, H-l'), 4.47 (d, 1 H, J l j 2 7.8 Hz, H-l), 4.19 (dd, 1 H, J5ax,5eq 12.1 Hz, H-5eq), 4.00-4.14 (m, 2 H, H-6a', H-6b') 3.94 (dd, 1 H, J 3 , 4 5.6 Hz, H-3), 3.89 (dd, 1 H, J 5 / i 6 a , 6.2 Hz, J5,t6V 7.3 Hz, H-5'), 3.59 (dd, 1 H, J5axA 7.0 Hz, H-5ax), 2.14, 2.10, 2.09, 2.05, 2.03, 1.97 (6 s, 18 H, Ac). Phenyl 2,3,4,6-tetra-0-(l-lzC-acetyl)-^-T>-mannopyranosyl-(l-^A)-2,3,6-tri-0-(l-nC-acetyl)-(3-D-glucopyranoside; X H NMR (400 MHz, CDC13): 5 5.47 (ddd, 1 H, J2>,AC 3.2 Hz, J2',3' 3.2 Hz, H-2'), 5.32 (m, 1 H, J4,>AC 1.9 Hz, H-4'), 5.20 (ddd, 1 H, J3tAc 3-18 Hz, J 3 ) 4 9.1 Hz, H-3), 5.13 (ddd, 1 H, J2Ac 3.41 Hz, J 2 , 3 10.0 Hz, H-2), 5.02 (ddd, 1 H, Jy>Ac 3.2 Hz, JZ,A, 6.4 Hz, H-3'), 4.66 (d, 1 H, JVt2, 0.5 Hz, H-l'), 4.30 (ddd, 1 H, J 6 M c 3.1 Hz, J 6 M a 12.4 Hz, H-6b), 4.26 (m, 1 H, H-6a), 4.10 (d, 1 H, J l j 2 7.1 Hz, H-l), 4.05-4.17 (m, 2 H, H-6a', H-6b'), 3.88 (dd, 1 H, J 4 , 5 9.6 Hz, H-4), 3.79 (ddd, 1 H, J 5 ) 6 a 2.44 Hz, J 5 ) 6 6 5.2 Hz, H-5), 3.62 (ddd, 1 H, J 5 , i 4 , 8.5 Hz, J 5 / ) 6 a , 2.2 Hz, Jg/.ef 5.0 Hz, H-5'), 2.17, 2.15, 2.14, 2.09, 2.05, 2.04, 1.98 (7 s, 21 H, Ac). 2,3,4,6-Tetra-0-(l-13C-acetyl)-(3-D-glucopyranosyl-(l -»• Z)-2,4,6-tri-0-(l-lzC-acetyl)-(3-D-glucopyranosyl-(l->6j-1,2,3,4-tetra-0-(l-13C-acetyl)-f3-D-glucopyranoside; : H NMR (400 MHz, CDC13): S 5.68 (dd, 1 H, J M c 3.3 Hz, J 1 > 2 8.2 Hz, H-l), 5.32 (ddd, 1 H, J 3 , A C 3.4 Hz, J 3 , 4 9.1 Hz, H-3), 5.16 (ddd, 1 H, J2,,Ac 3.4 Hz, J 2 „ > 3 „ 11.2 Hz, H-2"), 5.14 (ddd, 1 H, J3„Ac 3.3 Hz, H-3"), 5.14-5.03 (m, 2 H, H-4', H-4"), 5.05 (ddd, 1 H, J 2 > A c 1.5 Hz, 1 H, J 2 > 3 10.8 Hz, H-2), 5.04-4.89 (m, 1 H, H-4), 4.98 (ddd, 1 H, J2,tAc 0.1 Hz, J 2 , i 3 , 10.8 Hz, H-2'), 4.51 (ddd, 1 H, J 6 o , i 5 , 3.7 Hz, J6a,Ac 3.0 Hz, J 6 a , , 6 f / 12.4 Hz, H-6a'), 4.47 (d, 1 H, JVtV 7.8 Hz, H-l'), 4.33-4.20 (m, 3 H, H-6b', H-6a", C H A P T E R 4 Materials and Methods 87 H-6b"), 4.02 (d, 1 H, Jv,tV, 8.3 Hz, H-l"), 3.93-3.80 (m, 2 H, H-6a, H-5'), 3.81 (ddd, 1 H, J 5 ) 4 8.8 Hz, J5M 2.5 Hz, J5fib 3.7 Hz, H-5), 3.61 (m, 1H, H-6b), 3.57 (dd, 1 H, J 3 ' , 4 ' 9.0 Hz, H-3'). 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A P P E N D I X A Kinetic Analyses A . l Basic enzyme kinetics The velocity of a typical enzymatic reaction is described by the Michaelis-Menten equation: " = ¥TW W rs-m + p\ where v = the initial velocity of the reaction; a measure of the rate of depletion of substrate or formation of product [Ej] = the total amount of enzyme present (free and associated with substrate) [S] = the substrate concentration Kat = the catalytic constant K m = the Michaelis constant: that substrate concentration where v = Vmax/2 (half the maximal velocity) This expression is applicable when two conditions are met: (i) [E-r] <C [S], which is generally true since enzymes are usually very efficient catalysts; (ii) v is a true measure of the initial reaction velocity; that is, v is measured over a time period in which a small (<10%) amount of substrate has been depleted, or product, formed. 93 A P P E N D I X A Kinetic Analyses 94 2 (1/2)V, c > [ET][S] fe, kern [E-r] K m + [S] K m Substrate Concentration F I G U R E A . l : The Michaelis-Menten curve for a typical enzymatic reaction. The reverse of the reaction can be then be ignored and [S] will change approximately linearly with time. There are three distinct regions to the curve described by Equation A . l : (i) First order in substrate ([S] <C K m ) : most of the enzyme is not bound to substrate; therefore the amount of free enzyme ([E]) can be approximated as the total amount of enzyme present ([Ej])- Equation A . l then becomes: The parameter kcat/Km is the second order rate constant which reflects the first irre-versible step in reference to the free enzyme (E) and substrate (S). (ii) Zero order in substrate ([S] ~> K m ) : the initial reaction rate is independent of [S], and Equation A . l becomes: v = [E}[S]kcat (A.2) v = Vr max — (A.3) The catalytic constant, kcat, reflects the rate limiting step of catalysis. A P P E N D I X A Kinetic Analyses 95 (iii) In the region of the Michaelis-Menten curve where [S] ~ K m the reaction is between first and zero order with respect to [S]. The Michaelis-Menten expression is often graphed in double reciprocal form as a Lineweaver-Burk plot. This transformation results in a linear function: 1 v ^•r]kcat or substituting in Equation A.3: ET] kcat 1 Pi (A.4) 1 v Vm + Kr Vrr l_ (A.5) o > Slope = K,„ / Vmax Intercept = 1/ V max 1/Substrate Concentration F I G U R E A . 2 : The Lineweaver-Burk (double reciprocal) plot. A . 2 The Interpretation of kcat and kcat/Km The rate constants kcat and kcat/Krn reflect the free energy change associated with the rate determining step, and the first irreversible step of catalysis, respectively. This is demonstrated by the following argument. A P P E N D I X A Kinetic Analyses 96 Consider the following enzymatic reaction where the formation of E-S is the as-sociation step and the interconversion of E-S to E-P is the chemical step, followed by product release: k] kr ki E + S ====== E»S w E-P E + P k-\ k-2 The reaction coordinate diagram for this scheme is depicted in Figure A.3. It can be shown that the parameters kcat, Km and kcat/Km are defined as: k-> - k_2 + k2 + k3 < A ' 6 ) fc3(fc-l + k2) + fc-ifc-2 . K m = —TTi 71—7T~\— \AJ> kCat _ k\k2kz ( A Q\ ~ k3{k-i + k2) + k_xk_2 Consider a reaction with rapid, reversible association followed by a rate determin-ing chemical step, and product release (k2 <C k-i, k_2 <C fe, k2 <C A;3). Under these conditions, the kinetic parameters kcat and kcat/Km are reduced to: Kat = k2 (A.9) ^ - = (A.10) Km k-i The principles of transition state theory can be applied to Equations A.9 and A.10 to relate these rates constants to changes in free energy associated with the discrete A P P E N D I X A Kinetic Analyses 97 G E + P Reaction Coordinate F I G U R E A . 3 : Reaction coordinate diagram for a typical enzymatic reaction. En-ergy levels are arbitrary. steps of catalysis: kcat — Kr kT ^el-(Gls-GES)]/RT^ SSL = (p[-(GIS-Ge+s)}/RT) ( h kT (A.l l ) (A.12) where k = Boltzmann's constant T = temperature h = Planck's constant R = the gas constant APPENDIX A Kinetic Analyses 98 Both kcat and kcat/Km reflect the free energy change associated with the rate determining chemical step (the formation of E - P ) , but kcat uses the Michaelis-Menten complex ( E - S ) as a reference, and kcat/Km refers back to free enzyme ( E ) and substrate ( S ) . Now consider a reaction in which there is rapid, reversible association, fol-lowed by an irreversible chemical step and rate determining product release (k2 <C k-i, k3 <C k2, k-2 <^  k3). Under these conditions, kcat and kcat/Km reduce to: kCat = h (A.13) kcat k\k2 Again, from transition state theory, these expressions become: (A.14) k, cat v ca t fC ¥L (el-(GlP-GBP)]/RT^ ^ ( e - ^ / « r ) (A.16) In this case, kcat reflects the energy barrier for product release, yet kcat/Km again reflects that for the irreversible chemical step from free E and S . This argument demonstrates that kcat is the rate constant for the rate determining step, while kcat/Km is that for the first irreversible step of catalysis with respect to E and 5. In the hydrolysis of glycosides by BgaX, the formation of E - P and subsequent product release is analogous to glycosylation and deglycosylation respectively. Since kcat/Km reflects the free energy change associated with the first irreversible chemical step, it is the rate constant for glycosylation. However, the first order rate constant, kcat, may be associated with glycosylation or deglycosylation, depending on which step is rate limiting. A P P E N D I X A Kinetic Analyses 99 A . 3 Inactivation The inactivation of an enzyme ( E ) by a mechanism-based inactivator ( I X ) and subsequent reactivation by water is summarized by the following, where <C k2-k2 k? E + IX -. w E-IX ^ El J E + IOH I HX H 2 0 The kinetic expression for this scheme is analogous to the Michaelis-Menten equa-tion (A.l): • • im where ki = the inactivation rate constant Kj = the apparent dissociation constant between E and I X If [IX] ^> [ET] , then [IX] will change very little over the course of inactivation. This process will then be pseudo zero order with respect to [ IX] , and the reaction rate will be: v = k°bs[Ey] (A. 18) and Equation A.17 becomes The above equation is often presented in double reciprocal form as a linear function: = I 1 7ivT + r ( A - 2 ° ) kfs \ k i ) [IX] k. A P P E N D I X A Kinetic Analyses 100 Values of k°bs (the observed rate of inactivation) are obtained by fitting residual enzyme activity over time to a first order expression: £ = Afe-^+C (A.21) = the fractional enzymatic activity at time t = constants k"bs are then used with Equation A.20 to derive ki where V0 A, C The values thus obtained for and K,. A.4 The Determination of K j for a competitive inhibitor The association of a competitive inhibitor (P) with an enzyme and substrate is summarized by the following: E + S =^ = + P Ik E-S E + P k' k'_ E - P It can be shown that in the presence of P, the equation describing enzyme catalysis (A.l) becomes: [S] + K m ( l + [P]/K0 (A.22) where Kj = the apparent dissociation constant between E and P A P P E N D I X A Kinetic Analyses 101 1/Substrate Concentration F I G U R E A . 4 : The Lineweaver-Burk plots of enzyme catalysis in the presence (slopes) and absence (slope0) of P. The plots intercept on the y-axis, a pattern characteristic of competitive inhibition. or taking the reciprocal of the equation: 1 = 1 | 1 ( K , v Vmax [S] \Vm 1 + [P] Kv (A.23) Comparison of Equations A.5 and A.23 indicates that in the presence of P, the apparent Km of the substrate with the enzyme decreases by a factor of (1 + [P]/Kj), but Vmax is unchanged. Therefore, the slope of the Lineweaver-Burk plot of enzyme catalysis in the presence of P (sloped should differ from that in the absence of P (slope0) by a factor of (1 + [P]/K2), but both plots will still have the same y-intercept. That is: slop^ _ 1 [P] slope0 Ki or with rearranging Ki [P] slope, i slope0 (A.24) (A.25) A P P E N D I X A Kinetic Analyses 102 Therefore, an approximate value for the K j of a competitive inhibitor P can be obtained by the determination of the slopes of the double reciprocal plots defined by Equation A.5 in the presence and absence of P , and application of Equation A.25. A . 5 Protection from inactivation The illustration that an inhibitor (I) is active site-directed can be demonstrated by the protection of an enzyme from inactivation with a competitive inhibitor ( P ) as shown: E + I ^ w E- l + P - l E-P In the presence of P , it can be shown that the expression describing inactivation (Equation A.19) becomes: k t = , m ^ . . (A.26) K. ( l + Sg) + [I] where Kp = the apparent dissociation constant between E and P Combining Equations A.19 and A.26 and rearranging gives: Ks K i + [II (A.27) kf " K , ( l + g ) + [I] Therefore, if the ratio of the observed rates of enzyme inactivation in the presence (k°bs) and absence (kfs) of P agrees with Equation A.27, then it can be concluded that A P P E N D I X A Kinetic Analyses 103 inactivation is affected by the presence of a competitive inhibitor, and the inactivator under study is active site-directed. A . 6 Reactivation The reactivation of a glycosidase inhibited by a 2-deoxy-2-fluoro-glycoside (El) by transglycosylation to a suitable acceptor (A) is summarized in the following scheme: E l + A ^ E l - A k2 E + IA The observed increase in fractional enzyme activity (v/V0) follows a first order ex-pression: - = A i l - e (l _ e-*&c') + c (A.28) where Kllct — the observed rate of reactivation In order for adequate comparisons to be made between the reactivation profiles asso-ciated with the various acceptors studied in Chapter 3, the initial rate of reactivation tyreact-j w a g extrapolated from the above equation: yreact d (v dt \V0 t=o = -Ae AhfUe-^) lluobs \ \ ^react) 4=0 t=0 AUobs ^^react (A.29) Values of V™act were thus calculated and used to rank those glycosides that were identified as positively screened acceptors. A P P E N D I X A Kinetic Analyses 104 Time F I G U R E A.5: General plot of enzyme reactivation depicting the first order reac-tivation curve and the extrapolated slope of V^eact. APPENDIX B Graphical Representation of Data 1 General F I G U R E B . l : The determination of the molar absorptivity (e) of O pNP and • DNP with respect to pH. The pK a values for pNP and DNP were calculated to be 7.18 and 3.81 respectively from the equation e = Ll+L2{\0^n-pKa)^ 105 A P P E N D I X B Graphical Representation of Data 106 B.2 Enzyme kinetics The following seven plots depict the raw data for the initial reaction velocity versus substrate concentration for the hydrolysis of pNP- and DNP-glycosides by BgaX and fitting to t; = feg. 0 . 0 5 0.04 H 0 . 0 3 H ^ 0 . 0 2 0.01 0 . 0 0 0.0 0 .5 1.0 1.5 2 .0 2 .5 3.0 3 .5 [ p N P G a l ] (mM) 0.7 -i 0.6 -0 . 5 -'E 0 .4 -\ o 0 .3 -< > 0.2 -0.1 -0 .0 -0 .0 0 .2 0 .3 0 . 4 [ D N P G a l ] ( m M ) A P P E N D I X B Graphical Representation of Data 107 0.24 [ p N P - a - L - A r a ] (mM) A P P E N D I X B Graphical Representation of Data 108 0.008 0.006 § 0.004 < <3 0.002 0.000 1 2 3 [pNPGalNAc] (mM) 10 20 30 [pNPGlc] (mM) 40 < 0.015 0.012 0.009 0.006 0.003 0.000 5 10 15 [pNPXyi] (mM) 20 A P P E N D I X B Graphical Representation of Data 109 B.3 Enzyme reactivation The following plots depict the raw reactivation profiles (initial velocity versus time) in the presence of positively screened acceptors for the seven glycosidases studied after being corrected for enzyme death over time. The names of some of the acceptors have been abbreviated; consult the list of abbreviations for the full names of all glycosides. The profiles of each enzyme are separated into two or three plots for clarity. Note the scale of the y-axis (AAi00/min) may differ between graphs for a single enzyme to better accommodate the data. Data was fit to v = A 1^ — eA:°™ns'^ + C. Reactivation profiles (3) for BgaX: Note that the curve for pNPGlc was fit with the data up to t = 25 hr due to deviations from a first order expression at times greater than this. 0.6 0 20 40 60 80 Time (hr) A P P E N D I X B Graphical Representation of Data 110 A P P E N D I X B Graphical Representation of Data 111 Reactivation profiles (3) for BgaC: Since the activity of BgaC failed to approach a maximum value over the course of the experiment, each data set was fit to a linear expression to directly extrapolate yreact rpj^ v a m e Q£ yreact f o r m aitotriose (which fits to a full first order equation) was estimated from the data up to ca. 40 hr. The VJeact value for pNPGlc was estimated from the data up to 50 hr. Data collected after this time failed to correspond to a linear function. A P P E N D I X B Graphical Representation of Data 112 A P P E N D I X B Graphical Representation of Data 113 Reactivation profiles (2) for Man2A: The first order curve for pNPXyl and pNPMan were fit using the data up to ca. 2.5 hr. 0.25 n : 1 0 1 2 3 4 5 6 7 Time (hr) 0.12 0 1 2 3 4 5 6 7 Time (hr) A P P E N D I X B Graphical Representation of Data 114 Reactivation profiles (2) for Abg: 0.15 -c ' E 0.10 o o < > 0.05 0.00 - f — * 1 2 3 - 4 Time (hr) • D N P C e l l ° pNPGIc x C o n t r o l 5 c " E < < 0.15 H 0.10 0.05 0.00 i r 10 15 20. 25 Time (hr) A P P E N D I X B Graphical Representation of Data 115 Reactivation profiles (2) for CelB: 0.06 i i 1 0 5 10 15 20 25 Time (hr) Me—a—Cell G a l - / 3 - ( 1 - 3 ) - G l c - / S - S P h p N P X y l 2—deoxy—Gal c o n t r o l "i i i T 5 10 15 20 25 Time (hr) Me-p-CeW G a l - / S - ( l - 4 ) - G l c - / 3 - S P h PhGIc c o n t r o l A P P E N D I X B Graphical Representation of Data 116 Reactivation profiles (2) for Cex: Each data set was fit linearly to directly determine I / r e a c 4 . The slope associated with reactivation in the presence of pNPXyl was fit with the data up to ca. 30 hr. 0.0300 0 10 20 30 40 50 60 Time (hr) .000 H 1 1 1 1 1 1 0 10 20 30 40 50 60 Time (hr) A P P E N D I X B Graphical Representation of Data 117 Reactivation profiles (3) for HBG: 0.10-1 Time (hr) G a l - / S - ( 1 - 3 ) - G l c - / S - S P h f r u c t o s e A r t i c o n t r o l 0 50 100 150 200250300 Time (hr) A P P E N D I X B Graphical Representation of Data 118 0 . 0 8 Time (hr) 

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