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Purification and preliminary characterisation of β-glucosidase from Alcaligenes faecalis (ATCC 21400) Day, Anthony George 1985

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PURIFICATION AND PRELIMINARY CHARACTERISATION OF p -GLUCOSIDASE FROM ALCALIGENES FAECALIS (ATCC 21400) by ANTHONY GEORGE DAY B.Sc. (Hons.). Portsmouth Polytechnic, U.K., 1981 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1985 © Anthony George Day, 1.985 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date ! £ / % / £ > _ i i To my Mother S.J. Day i i i A b s t r a c t A B - g l u c o s i d a s e was i s o l a t e d from A. f a e c a l i s and p u r i f i e d 880 f o l d by a c o m b i n a t i o n of c l a s s i c a l and medium p r e s s u r e c h r o m a t o g r a p h i c t e c h n i q u e s t o a s p e c i f i c a c t i v i t y of 31.6 u n i t s / m g . The p r o t e i n was homo-geneous by t h e c r i t e r i a o f SDS-PAGE and g e l chromatography. The s u b - u n i t m o l e c u l a r w e i g h t was d e t e r m i n e d t o be 51,000 by SDS-PAGE. The a p p a r e n t o l i g o m e r i c m o l e c u l a r w e i g h t was d e t e r m i n e d t o be 75,000 by Superose g e l chromatography and 98,000 by Waters 1-250 g e l chromatography, s u g g e s t i n g t h a t the enzyme i s a d i m e r . The enzyme was shown t o be a r e t a i n i n g ( 3 - g l u c o s i d a s e w i t h e x o -g l u c a n a s e a c t i v i t y o n l y . The k i n e t i c p a rameters of a number of s u b s t r a t e s and i n h i b i t o r s were d e t e r m i n e d a l l o w i n g d e d u c t i o n s t o be made about the n a t u r e of the a c t i v e s i t e and c a t a l y t i c mechanism. The Km's d e t e r m i n e d f o r c e l l o b i o s e and PNPG were low f o r a b a c t e r i a l B - g l u c o s i d a s e , 0.70 mM and 0.083 mM r e s p e c t i v e l y . I n the c e l l o d e x t r i n s e r i e s , c e l l o b i o s e t o c e l l o p e n t a o s e , the enzyme was most e f f i c i e n t (as d e f i n e d by Vm/Km) w i t h c e l l o t r i o s e as s u b s t r a t e . I n common w i t h o t h e r c e l l u l o l y t i c 8 - g l u c o s i d a s e s , the g l y c o n e s i t e showed a h i g h s p e c i f i c i t y f o r g l u c o s e ( a l t h o u g h i t would t o l e r a t e some m o d i f i c a t i o n s ) and poor s p e c i f i c i t y a t the a g l y c o n e s i t e . C a t a l y t i c a c t i -v i t y was ( u n u s u a l l y ) o b s e r v e d w i t h p - n i t r o p h e n y l - 8 - D - m a n n o p y r a n o s i d e as s u b s t r a t e . A c t i v a t i o n e n e r g i e s were d e t e r m i n e d by means of A r r h e n i u s p l o t s . i v Table of Contents Page Dedication i i Abstract i i i Table of Contents i v L i s t of Tables v i i L i s t of Figures v i i i Acknowledgements x i i I. Introduction 1 1.1 General 2 1.2 Mechanism of Glycosidase Cat a l y s i s 4 1.3 Enzyme I s o l a t i o n 24 1.4 Previous Studies on B-Glucosidase from A. f a e c a l i s . . 34 I I . Experimental » 35 Abbreviations Used 36 I I . 1 Synthesis 37 11.1.1 General Methods 37 11.1.2 p-Nitrophenyl - 8-D-cellobioside 39 11.1.3 p-Nitrophenyl - 8-D-glucopyranoside . . 40 11.1.4 Isopropyl - 8-D-l-thioglucopyranoside 42 11.1.5 8-D-Glucopyranosyl Pyridinium Bromide 44 11.1.6 8-D-Glucopyranosyl Azide 45 11.1.7 8-D-Glucopyranosyl Fluoride 45 11.1 .8 8-D-Glucopyranosylamine 46 V Page II.1.9 2,4-Dinitrophenyl-l-thio-p-D-glucopyranoside . 47 11.2 Growth of A. f a e c a l i s , ATCC, 21400, and Preparation of C e l l Free Extract 49 11.2.1 General Methods 49 11.2.2 Growth of A. f a e c a l i s 49 11.2.3 {3-Glucosidase Induction Studies 55 11.2.4 Preparation of C e l l Free Extract from A. f a e c a l i s ; C e l l Breakage 56 11.3 P u r i f i c a t i o n and I s o l a t i o n of B-Glucosidase from A. f a e c a l i s and Related Det a i l s 62 11.3.1 General Methods 62 11.3.2 P u r i f i c a t i o n and I s o l a t i o n of 8-Glucosidase from A. f a e c a l i s 64 11.3.3 Method Development 77 11.3.4 Enzyme Storage 79 11.3.5 Unsuccessful P u r i f i c a t i o n Techniques Attempted 80 11.4 Characterisation of 8-Glucosidase from A. f a e c a l i s . . 84 11.4.1 General Methods 84 11.4.2 Molecular Weight Determination 86 11.4.3 Kin e t i c s 89 11.4.4 Miscellaneous Characterisation 97 11.5 Treatment of Data 100 v i Page III Results and Discussion 103 111.1 P u r i f i c a t i o n (and Comparison with Previous Work) 104 111.2 S t a b i l i t y (and Comparison with Previous Work) 106 111.3 Molecular Weight (and Comparison with Previous Work) 109 111.4 Anomeric Configuration of I n i t i a l Products . . I l l 111.5 K i n e t i c and Thermodynamic Data 112 111.5.1 Results 112 111.5.2 Comparis on with Previous Work . . . . 115 111.5.3 Substrates; Implications for the Active Sites and Mechanism 116 111.5.4 I n h i b i t o r s ; Implications for the Active Site and Mechanism 126 I I I . 6 Conclusion 129 Bibliography 130 Appendix 1; Basic Enzyme Ki n e t i c s 137 Appendix 2; Program for Apple l i e Computer Used to Determine Km and Vm 143 Appendix 3; Graphical Representation of Kinetic Data 149 1. Substrates 150 2. Inhibitors 158 3. Substrate I n h i b i t i o n 161 Addendum; Molecular Weight Determination on Waters 1-250 Column 162 v i i L i s t of Tables Page I I n h i b i t i o n Constants of some Cationic and Neutral Inhibitors with B-Glucosidase 13 II Functional Groups Used i n Ion-Exchange Materials 28 III V a r i a t i o n of C e l l Density and 8-Glucosidase A c t i v i t y with Time 54 IV Comparison of C e l l Breakage Techniques 59 V P u r i f i c a t i o n of 8-Glucosidase from A. f a e c a l i s 74 VI Buffers used i n Determination of Conditions for Mono Q Chromatography 79 VII Molecular Weight Markers for SDS-PAGE 86 VIII Molecular Weight Markers for Superose 12 Gel Chromatography 87 IX 8-Glucosidase Substrates 90 X Ki n e t i c Parameters of 8-Glucosidase with Several Substrates at 37°C and pH 6.8 112 XI Ki n e t i c Parameters of 8-Glucosidase with Several Inh i b i t o r s at 37°C and pH 6.8 113 XII Molecular Weight Markers for Waters 1-250 Gel Chromatography 162 v i i i L i s t of Figures Page Figure 1 Cleavage of Glucoside Bond 2 Figure 2 Retaining and Inverting Glucosidases 4 Figure 3 Proposed Glycosidase Mechanism 5 Figure 4 Substrate for Lysozyme 7 Figure 5 Oxocarbonium Ion T r a n s i t i o n State 8 Figure 6 Minimal K i n e t i c Mechanism for Galactoside Hydrolysis . 9 Figure 7 Reaction of Conduritol B cis-Epoxide with B-Glucosidase 11 Figure 8 Epoxide Inhibitors of Glucosidases 12 Figure 9 I n h i b i t i o n of 8-Glucosidase by D-Glucono-6-lactone . • 14 Figure 10 Ki Ratios of Some I s o s t e r i c Cationic and Neutral I n h i b i t o r s 15 Figure 11 Proposed 8-Glucosidase T r a n s i t i o n State 16 Figure 12 Unusual Glycosidase Substrates 17 Figure 13 Hydration of Glucal by 8-Glucosidase 18 Figure 14 Plot of Log(kcat./Km) vs. pH for 8-Glucosidase . . . . 19 Figure 15 Proposed C a t a l y t i c Mechanism of 8-Galactosidase from E. c o l i 21 Figure 16 Nojirimycin and Acarbose 22 Figure 17 Plot of Log ( s o l u b i l i t y ) vs. Ionic Strength for Haemoglobin 25 Figure 18 P u r i f i c a t i o n of Staphylococcal Nuclease by A f f i n i t y Adsorption Chromatography 30 i x Page Figure 19 Va r i a t i o n of C e l l Density, S p e c i f i c A c t i v i t y and B-Glucosidase A c t i v i t y with Time 54 Figure 20 Flow Chart for the I s o l a t i o n and P u r i f i c a t i o n of 8-Glucosidase from A. f a e c a l i s 67 Figure 21 P r o t e i n / A c t i v i t y P r o f i l e for DE-52 Chromatography step. 70 Figure 22 P r o t e i n / A c t i v i t y P r o f i l e for F i r s t S-200 Chromatography Step 70 Figure 23 P r o t e i n / A c t i v i t y P r o f i l e for Mono Q (Phosphate) Chromatography Step 71 Figure 24 P r o t e i n / A c t i v i t y P r o f i l e for Mono Q (Triethanolamine) Chromatography Step 71 Figure 25 Protein A c t i v i t y P r o f i l e for Second S-200 Chromatography Step 72 Figure 26 SDS-PAGE Gel of Pure and P a r t i a l l y Pure 8-Glucosidase . 75 Figure 27 A n a l y t i c a l Gel Chromatography of Pure B-Glucosidase on Superose 12 Column 75 Figure 28 UV Spectra of A f f i n i t y E l u t i o n Buffer Before and After Passage Through a DE-52 Column Containing B-Glucosidase 83 Figure 29 Vari a t i o n of PNP Ex t i n c t i o n C o e f f i c i e n t with Temperature at pH 6.8 85 X Page Figure 30 Plot of Log (molecular weight) Against Rf for SDS-PAGE. 88 Figure 31 Logarithmic Plot of Molecular Weight Against Rf for Superose 12 Gel Chromatography 88 Figure 32 C a l i b r a t i o n Curve for Fluoride Electrode 94 Figure 33 S p e c i f i c Optical Rotational Changes During Hydrolysis by 8-Glucosidase I l l Figure 34 Arrhenius Plots of In Vm and In Vm/Km against 1/T . . . 114 Figure 35 I n i t i a l Rate of Hydrolysis of p-Nitrophenyl-B-D-c e l l o b i o s i d e at a Number of Concentrations 117 Figure 36 Schematic Representation of p-Glucosidase Binding S i t e . 120 Figure 37 Hypothetical 8-Glucosidase K i n e t i c Mechanism 122 Figure 38 Hypothetical 8-Glucosidase Chemical Mechanism 123 Figure 39 Kinetic Mechanism for P a r t i a l l y Non-Competitive I n h i b i t i o n 127 Figure 40 Schematic Model for P a r t i a l l y Non-Competitive I n h i b i t i o n 128 Graphical Representation of Kinetic Data; Substrate Plots of:-Figure 41 PNPG 150 Figure 42 DNPG 150 Figure 43 DNPTG 151 Figure 44 8-D-Glucopyranosyl Azide ' 151 x i Page Figure 45 PNPMan 152 Figure 46 PNPGal 152 Figure 47 8-D-glucopyranosyl Fluoride 153 Figure 48 PY-G 153 Figure 49 Gentiobiose 154 Figure 50 Sucrose 154 Figure 51 S a l i c i n 155 Figure 52 Lactose . .' 155 Figure 53 Cellobiose 156 Figure 54 C e l l o t r i o s e 156 Figure 55 Cellotetraose 157 Figure 56 Cellopentaose 157 Inhibitor Plots of:-Figure 57 p-Nitrophenyl-oc-D-glucopyranoside 158 Figure 58 p-Nitrophenyl-B-D-cellobioside 158 Figure 59 8-D-Glucosylamine 159 Figure 60 D-Glucono-5-lactone 159 Figure 61 B-D-Glucopyranose 160 Figure 62 8-D-Glucopyranose, Secondary Replots 160 Substrate I n h i b i t i o n plots of:-Figure 63 PNPG 161 Figure 64 PNPGal 161 Figure 65 Plot of Log (Molecular Weight) Against Rf for Waters 1-250 gel chromatography 163 x i i Acknowledgements I would l i k e to thank my supervisor, Professor S.G. Withers for h i s guidance and unstinting support throughout the period of this work, and for his help i n the preparation of th i s t h e s i s . I should also l i k e to thank N e i l Gilkes, Warren Wakarchuk (Microbiology Dept., UBC) and Dr. Paul Bird (Chemistry Dept., UBC), and facul t y and graduate students of the Chemistry Department too numerous to mention for both h e l p f u l discussions and chemical 'loans'. Professor Chris Orvig, Dr. Pat MacNeil and Jane Clark also deserve thanks for proofreading t h i s manuscript; any remaining mistakes i n the thesis almost c e r t a i n l y occured a f t e r they had handed back the manuscript. I am g r a t e f u l for the s k i l l e d and prompt service supplied by the technical s t a f f of the Department, i n p a r t i c u l a r to Mr. G. Hewitt for his help and guidance i n mi c r o b i o l o g i c a l techniques, and to Mr. P. Borda for his accurate analyses. Thanks are also due to the Chemistry Department for the support and f a c i l i t i e s that allowed me to carry out work that I found both f a s c i n a t i n g and stimulating. F i n a l l y , no acknowledgement would be complete without thanking St. Jude for coming through when he was needed most, and the student radio s t a t i o n , CITR FM 102, for helping me to keep my sanity when he took his time i n getting there. 1 CHAPTER I Introduction 2 1.1 General B-Glucosidase (B-D-glucoside glucohydrolase, 3.2.1.21) i s an enzyme which catalyses the cleavage of B-glucosidic bonds, F i g . 1. Figure 1: Cleavage of Glucoside Bond. The natural substrates for the enzyme have an aglycone (R) which may be either a r y l or g l y c o s y l . 1 8-Glucosidases are ubiquitous i n nature and have been i s o l a t e d from fungal, b a c t e r i a l , yeast, plant and animal sources, including man. 1* 2 They are often found associated with, or as part of, the c e l l u l a s e complex. This i s an enzyme complex which catalyses the hydrolysis of c e l l u l o s e , a B-l,4-linked polymer of glucose, to glucose u n i t s . It i s known to be composed of at l e a s t three or four d i f f e r e n t classes of enzyme,1 v i z . endo -8-1,4-glucanase,exo-p-l,4-glucanase, cellobiohydrolase 3 (which is also an exo-glucanase) and 8-glucosidase. Endo-B-1,4-glucanase catalyses the hydrolysis of the glucosidic bonds of cellulose at random internal points increasing the number of chain ends. Exo-8-1,4-glucanase and cellobiohydrolase catalyse the cleavage of oligosaccharide and cellobiose units, respectively, from the non-reducing ends of the polymer chains. B-Glucosidase catalyses the hydrolysis of cellobiose into glucose. Cellobiose inhibits both exo- and endo-gluconases and i ts hydrolysis by B-glucosidase, in addition to providing the last step in glucose production, is important to the efficiency of the cellulase complex. Cellulose is a major component of industr ia l , municipal and agricultural waste. Recycling by degradation to glucose (and subsequent fermentation to ethanol) has been of increasing economic and environmental interest over the past one or two decades. Endeavours to clone a cellulase complex from the bacterium Cellulomonas fimi into Escherichia c o l i have been embarked upon with a view to achieving expression of the complex at higher levels, thus producing a very efficient means of degrading cel lulose. However, there have been problems with the cloning of the B-glucosidase component, and the co-cloning of a 8-glucosidase from Agrobacter faecalis has been, and is s t i l l being, attempted. 3 Screening procedures used in this attempt would be fac i l i tated by the use of pure protein and i t was felt that a preliminary characterization of the purified enzyme, both physically and mechanistically would be useful. A discussion of some of the glycosidase mechanism l iterature follows. 4 1 . 2 Mechanism of Glycosidase Catalysis In a l l of the glycosidases so far studied, bond cleavage has been shown (by 1 8 0 labelling) to occur between the anomeric carbon and the glycosidic oxygen.** Glycosidases may be divided into two categories, v i z . those proceeding with retention of configuration at the anomeric centre and those proceeding with inversion, Fig. 2 . Figure 2 : Retaining and Inverting Glucosidases. Most mechanistic studies have been carried out on 'retaining' glycosidases and they are assumed to have common mechanistic features. This discussion w i l l be limited to this class of enzymes. Some of the processes that have been suggested^ to be mechanis-t i c a l l y important to catalysis by glycosidases are: a) intra-complex 5 general acid c a t a l y s i s , b) lntra-complex n u c l e o p h i l l c c a t a l y s i s and c) e l e c t r o s t a t i c s t a b i l i s a t i o n of an oxocarbonium ion intermediate. These are i l l u s t r a t e d below, F i g . 3. Figure 3: Proposed Glycosidase Mechanism. Other factors that may be important are: d) conformational d i s t o r t i o n of the substrate, i . e . some of the binding energy i s used to d i s t o r t or to 6 ' p u l l ' the ground state substrate towards a t r a n s i t i o n state conformation, and e) a microscopic medium e f f e c t i n which any of the above might function more e f f i c i e n t l y i n the enzyme substrate complex than i n an aqueous environment. The l a t t e r hypothesis, whilst being implied from the X-ray c r y s t a l l o g r a p h i c data on the (e.g. hydrophobic) amino acid residues found at the active s i t e s of a number of enzymes, i s d i f f i c u l t i f not impossible to prove or disprove unequivocally and w i l l not be discussed further. The only glycosidase that has been well characterised by means of X-ray crystallography to date i s hen eggwhite lysozyme 6 >7 and much of the evidence for the above postulated mechanisms has come from the data on t h i s enzyme. The natural substrate for t h i s enzyme i s a B-1,4 linked co-polymer of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM), F i g . 4. Studies involving the b u i l d i n g of substrate models into the active s i t e of the enzyme (as determined c r y s t a l l o g r a p h i c a l l y ) suggest that i t consists of six subsites (designated A-F) binding alternate NAG, NAM moieties. Cleavage takes place between the D and E subsites, with the D subsite binding a h a l f - c h a i r conformation of i t s sugar better than the preferred ground state conformation, a f u l l - c h a i r . 5 > 6 The amino acid residues thought to be important i n c a t a l y s i s are glutamic acid 35 and aspartate 52 5> 6 since they are located i n the active s i t e on opposite sides of the g l y c o s i d i c bond that i s cleaved. This evidence implies that the substrate i s bound at the active s i t e and d i s t o r t e d toward the 7 Figure 4: Substrate for Lysozyme. conformation of an oxocarbonium ion l i k e t r a n s i t i o n state. However i t i s not clear whether the enzyme a c t u a l l y d i s t o r t s the substrate ground state or just ' p u l l s ' i t toward the t r a n s i t i o n state by binding the t r a n s i t i o n state better than the undistorted substrate, 8 F i g . 5. The g l y c o s i d i c bond i s then cleaved with general acid c a t a l y t i c assistance from glutamic acid 35, accompanied either by d i r e c t n u c l e o p h i l i c p a r t i c i p a t i o n , or less d i r e c t l y , by oxocarbonium ion s t a b i l i s a t i o n by aspartate 52 as shown i n F i g . 3. Crystallographic studies should, however, be interpreted with caution. It had been suggested 6 from these studies that s t e r i c i n t e r -8 actions between the C g 0 H and a group i n the D subsite provide the main Figure 5 : Oxocarbonium Ion T r a n s i t i o n State. source of d i s t o r t i o n towards the h a l f - c h a i r sugar conformation i n lysozyme. However work by Capon 9 has shown that lysozyme i s completely i n a c t i v e with substrate analogues having xylo, 6-chloro, 6-fluoro and 6-deoxy sub s t i t u t i o n s , suggesting a more subtle e f f e c t than simple s t e r i c i n t e r a c t i o n s . The involvement of some kind of glycosyl-enzyme intermediate i n the glycosidase c a t a l y t i c mechanism has been demonstrated i n a number of ways i n d i f f e r e n t glycosidases. Nucleophilic competition studies with 8 - S a l a c t o s l d a s e from E. c o l i i nvolving the use of ethanol and methanol as competing nucleophiles (against water) have been c a r r i e d o u t . 1 0 The product r a t i o measured (alkyl-galactoside/galactose) was found to be independent of the aglycone leaving group for a series of a r y l galactosides. This implies a common intermediate (E»G) i n the s o l v o l y s i s of these substrates, F i g . 6. 9 HOR' E *GOR' k +i HOR E • GOR E-GOR k-1 E • GOH Figure 6: Minimal K i n e t i c Mechanism for Galactoside Hydrolysis. Nucleophilic competition studies have also been used i n the same system to show that the rate l i m i t i n g step i s degalactosylation (k+3,k+4) for 'fast' s u b s t r a t e s 1 1 by measuring the increased rate of s o l v o l y s i s observed i n the presence of competing nucleophiles. 'Cryosolvents' (eg. 50% aqueous DMSO) at low te m p e r a t u r e 1 2 * 1 3 and 'stopped flow* apparatus at 20°C 1 1 + have been used to investigate the hydrolysis of p-nitrophenyl-8-D-glucopyranoside catalysed by 8-glucosidase from sweet almonds under conditions i n which deglucosylation was expected to be rate l i m i t i n g . An i n i t i a l rapid release of p-nitrophenol (the f i r s t product) was observed, followed by a decrease i n rate to the steady state rate of p-nitrophenol production. The i n i t i a l 'burst' i s assumed to be due to rate l i m i t i n g cleavage of the g l u c o s i d i c bond (k+2) equivalent to one enzyme turnover. The subsequent steady state rate i s due to rate l i m i t i n g deglucosylation (k+4). The a c t i v a t i o n energies, for both bond 10 cleavage and deglucosylation, calculated from low temperature pre-steady state k i n e t i c s , were consistent with those calculated from steady state k i n e t i c s at higher temperatures. 1 3 Inhibitors have been u t i l i s e d extensively to probe the c a t a l y t i c mechanism of g l y c o s i d a s e s . 1 5 There are two main classes of i n h i b i t o r of importance i n mechanistic studies, v i z . covalent ( i r r e v e r s i b l e ) and non-covalent competitive ( r e v e r s i b l e ) i n h i b i t o r s . Within the former (covalent) class are two major types of i n h i b i t o r : the active s i t e directed and the suicide i n h i b i t o r s . While both resemble the natural substrate, the former are i n t r i n s i c a l l y r eactive, whilst the l a t t e r remain unreactive u n t i l activated by the action of c a t a l y t i c groups at the active s i t e . Both classes, by v i r t u e of t h e i r s t r u c t u r a l resemblance to the substrate, become covalently attached at the active s i t e . Suicide i n h i b i t o r s however, due to t h e i r requirement for c a t a l y t i c a c t i v a t i o n , become attached at the active s i t e almost e x c l u s i v e l y . In order that a c a t a l y t i c a l l y important glycosidase residue be l a b e l l e d by a covalent i n h i b i t o r the reactive part of the molecule should be d i r e c t l y attached to the anomeric carbon. 1 6 Epoxide derivatives have been used e x t e n s i v e l y . 1 5 Conduritol B c i s - ( F i g . 7) and trans-epoxides i n h i b i t (8) and (a)-glucosidases r e s p e c t i v e l y ; incorporation of one mole of i n h i b i t o r per mole of enzyme active s i t e s leading to complete i n a c t i v a t i o n . The i n h i b i t o r i s released from the enzyme by the action of hydroxylamine and the stereochemistry of the released products i s consistent with the proposed mechanism, F i g . 7. 1 5 11 a-Glucosidases are Inhibited 50-200 times more slowly (by the trans epoxide) than are B-glucosidases (by the c i s epoxide) and this may be because the former do not go through a t r a n s - d i a x i a l r i n g opening i n the proposed mechanism. 1 5 nmTTT m i n i P H — Figure 7: Reaction of Conduritol B cis-Epoxide with B-Glucosidase. Glucosidases that have been l a b e l l e d with radioactive conduritol B epoxides have been subjected to p r o t e o l y t i c hydrolysis and the peptides containing r a d i o a c t i v i t y sequenced. 1 5 In a l l cases so far studied i t i s an aspartate residue that i s l a b e l l e d . 1 5 A s h i f t i n the epoxide function (eg. F i g . 8) diminishes, or even destroys i n h i b i t o r y a c t i v i t y . 1 5 This suggests a f a i r l y r i g i d o r i e n t a t i o n of c a t a l y t i c groups at the active s i t e . 12 Figure 8 : Epoxide Inhibitors of Glucosidases. Isothiocyanate (-N=C=S), N-bromoacetyl (-NHCOCH2Br) and triazene (-CH2NHN=N-0-NO2) glycoside derivatives have also been used as covalent i n h i b i t o r s although the r e s u l t s are less well d e f i n e d . 1 5 The use of competitive i n h i b i t o r s to probe the mechanism of glycosidases i s well i l l u s t r a t e d by the work c a r r i e d out on 8-glucosidase from A s p e r g i l l u s wentii by Legler et a l . 1 7 The Ki values determined for a Ki i s a d i s s o c i a t i o n constant for an i n h i b i t o r which i s determined k i n e t i c a l l y by quantifying i t s i n h i b i t i o n of the normal r e a c t i o n . It should also be noted that Km i s an apparent d i s s o c i a t i o n constant for the substrate and kcat i s the maximal reaction rate at saturating substrate concentration. These terms and t h e i r derivation are discussed more f u l l y i n Appendix 1. 13 Table I x / : I n h i b i t i o n Constants of some Catlonlc and Neutral  Inhibitors with 8-Glucosidase. Inhibit o r K ± (mM) -AAG° (kJ/mole) B-glucose 2.8 -8-2-amino-2-deoxyglucose 18 0 B-glucosylamine 0.0016 18.6 N-bromoacetyl -B-glucosylamine 0.25 6.6 D-glucono-6-lactone 0.0096 14.6 5-amino-5-deoxy-D-gluconolactam 0.036 11.2 8-glucosylbenzene 96 -N - B-glucosylpyridinium ion 0.30 14 N - B - g l u c o s y l p i p e r i d i n e 0.091 17 N-8-glucosylimidazole 5.9 7 series of c a t i o n i c and neutral i n h i b i t o r s are shown (Table I ) . D-Glucono-6-lactone has an ad d i t i o n a l binding energy, compared to glucose, of 14.6kJ mole - 1. This Increase In binding energy was at t r i b u t e d to t h i s compound acting as a 't r a n s i t i o n - s t a t e * analogue. Its preferred ground state conformation, a h a l f - c h a i r , i s analogous to that expected for an oxocarbonium ion l i k e t r a n s i t i o n state. Another explanation of this tight binding i s a re v e r s i b l e reaction with a c a t a l y t i c group i n the 14 active s i t e of the enzyme. 1 8 Both of these p o s s i b i l i t i e s are i l l u s t r a t e d below, F i g . 9. Figure 9: I n h i b i t i o n of 8-Glucosidase by D-Glucono-6-lactone. The t i g h t binding of the i n t r i n s i c a l l y less reactive 5-amino-5-deoxy-D-gluconolactam favours the former explanation. However B-glucosidase from sweet almonds, whilst binding the lactone t i g h t l y , binds the lactam only s l i g h t l y better than 8-D-glucose, and i n t h i s case the l a t t e r explanation i s f a v o u r e d . 1 8 Inhibitors bearing a p o s i t i v e charge on the exocyclic atom attached to the anomeric carbon were found to bind t i g h t e r than analogous neutral i n h i b i t o r s . This i s i l l u s t r a t e d by the Ki r a t i o s shown below, F i g . 10. Support for the hypothesis that this increase i n binding energy 15 B-D-glucosyl benzene: 8-D-glucosyl 8-D-glucose: 8-D-glucosylammonium ion pyridinium ion K ± ° / K + = 320 K ± ° / K + = 4000 Figure 10: Ki Ratios of Some I s o s t e r i c Cationic and Neutral I n h i b i t o r s . i s due to the influence of a single point negative charge (rather than the contribution of a number of charges) i s found i n the increase i n binding energy (AAG°, Table I) observed for the 8-D-glucosyliraidazolium ion. The increase observed i s that anticipated for a half charge on the nitrogen attached to the anomeric centre with no contribution from the charge on N-3. Furthermore, the 2-amino-2-deoxyglucosyl cation binds no more t i g h t l y than glucose, implying that the charge i s located i n the d i r e c t 16 v i c i n i t y of the anomeric carbon. From these, and other data, the following t r a n s i t i o n state was suggested, F i g . 11. Figure 11: Proposed 8-Glucosidase T r a n s i t i o n State. Summarising the Implications of the evidence discussed so f a r , i t seems that for r e t a i n i n g glycosidases an enzyme intermediate i s involved, most l i k e l y having some degree of oxocarbonium ion character. Carboxylate bearing groups are probably intimately involved with c a t a l y t i c a c t i v i t y and the c a t a l y t i c groups are f a i r l y r i g i d l y placed with respect to the bond to be cleaved. In short then, this evidence i s consistent with the mechanism proposed i n F i g . 3. 17 Two unusual classes of substrate hydrated by some glycosidases are g l y c a l s 1 9 » 2 0 and h e p t e n i t o l s 2 1 , F i g . 12. HePtenitoi Figure 12: Unusual Glycosidase Substrates. Glycals, by v i r t u e of t h e i r s p 2 h y b r i d i z a t i o n at the anomeric center, and therefore h a l f - c h a i r conformation, were o r i g i n a l l y thought to be ' t r a n s i t i o n state analogues' 1 5. However the tight binding and time dependent (on the order of minutes) i n h i b i t i o n sometimes observed have been at t r i b u t e d to enzymatically catalysed hydration i n which both enzyme gl y c o s y l a t i o n and deglycosylation are sometimes s l o w 1 9 » 2 0 . Denaturation and subsequent p r o t e o l y t i c digestion of 6-glucosidase from A. wentii which had been incubated with radio l a b e l l e d D-glucal yielded a r a d i o - l a b e l l e d p e p t i d e 2 0 . Amino acid analysis showed that the glucosyl moiety was attached to the same aspartate residue as had been l a b e l l e d by conduritol 18 B epoxide. When the reaction was performed i n D 20, the stereochemistry of the products could be determined and a mechanism proposed, F i g . 13. 1 5 Figure 13: Hydration of Glucal by B-Glucosidase. The less reactive 5-thio-D-glucal i s not hydrated and binds only weakly. 2 2 Glycals are therefore not t r a n s i t i o n state analogues. 2,6-Anhydro-l-deoxy-D-gluco heptenitol i s hydrated to 1-deoxy-D-gluco heptulose, F i g . 12, by both a- and B-glucosidases with 'retention'. 1 5» 2 1 The reaction i s proposed to go through the analogous c a t a l y t i c pathway (to the normal r e a c t i o n ) . 1 5 * 2 1 Deoxy analogues of glucosyl substrates have been used to probe the mechanism of B - g l u c o s i d a s e s 2 3 » 21* > 2 5 Such subst i t u t i o n s , in addition 19 to a l t e r i n g Km, often lead to a decrease i n kcat. The most marked decrease i n kcat. (up to 10 6 times) occurs when a 2-deoxy s u b s t i t u t i o n i s made. 2 3 These e f f e c t s have been var i o u s l y attributed to: poor substrate deformation on binding (2-deoxy), 2 3 lack of 'induced f i t ' 2 3 ' 2 4 , or incorrect alignment of c a t a l y t i c groups due to increased r o t a t i o n a l freedom of the glucosyl moiety. 2 1 +» 2 5 . The v a r i a t i o n of kcat. ( r e f l e c t i n g the rate determining step) and kcat./Km ( r e f l e c t i n g the f i r s t i r r e v e r s i b l e step, see appendix I) with pH i s often measured. For glycosidases the plot i s often ' b e l l ' shaped, eg. F i g . 14 2 9, and t h i s i s taken to imply that one protonated and one deprotonated residue are necessary for c a t a l y s i s . Attempts are often 2 6 -2 * --1 1 1 1 L_ SO SS 60 65 70 pH Figure 14 2 9: Plot of Log, Q(kcat/Km) vs. pH for 8-Glucosidase. The hypothesis of 'induced f i t ' of a substrate was proposed by Koshland 2° to explain enzyme s p e c i f i c i t y . The binding of the correct substrate i s proposed to induce a conformational change i n the enzyme which brings amino acid residues at the active s i t e into the correct alignment for c a t a l y t i c a c t i v i t y . Evidence for t h i s hypothesis was f i r s t found for a-amylase. 2 7 This hypothesis has been d i s p u t e d 2 8 20 made to extract the pKa's of these residues B*29 f r 0 m the points of i n f l e c t i o n of the p l o t s . However Knowles, i n an aut h o r i t a t i v e review of the s u b j e c t 3 0 , has pointed out the f o l l y of any s i m p l i s t i c Interpretation of pH p r o f i l e s without corroborative data from other sources. Some work which manages to avoid most of these p i t f a l l s i s that of Legler et a l . 3 1 A l l of the above mentioned work i s consistent with the mechanism proposed, F i g . 3. 3 1 However l i t t l e has been said about the nature of the enzyme intermediate and i t s conversion to products. C l e a r l y , simple S^l hete r o l y s i s producing an i s o l a t e d oxocarbonium ion i s u n l i k e l y since the predicted l i f e t i m e of such an intermediate i s of the order of 10" 1 1 -1 0 - 1 5 seconds. 3 2 Kinetic studies have been c a r r i e d out on B-galactosidase from E. c o l i 1 8 > 3 3 - 3 7 with both galactosyl pyridinium s a l t s (which cannot undergo general acid catalysed hydrolysis) and a r y l galactosides as substrates. These studies involved c o r r e l a t i o n of kcat. with pKa of the aglycone leaving group and determination of both a-deuterium and 1 8 0 k i n e t i c isotope e f f e c t s ( r e f l e c t i n g bond order and bond s c i s s i o n i n the t r a n s i t i o n s t a t e ) . These studies gave r i s e to the following proposed mechanism, F i g . 15. The existence of the ion-pair equilibrium represented by K. has been well reviewed. 1 + 0 21 Figure 15: Proposed Catalytic Mechanism of B-Galactosidase from E. c o l i . The results of the 1 8 0 KIE studies 3 9 and the pKa/kcat. s tudies 1 8 suggest different rate l imit ing steps. It is possible that this apparent contradiction may be resolved in the light of recent work 3 8 on the 22 s p e c i f i c i t y of the aglycone binding s i t e . This suggests that i n some cases release of aglycone may be rate l i m i t i n g . " 4 0 It i s g r a t i f y i n g to f i n d that some na t u r a l l y occurring glycosidase  i n h i b i t o r s i s o l a t e d from b a c t e r i a l s o u r c e s , 1 5 F i g . 16, have s t r u c t u r a l c h a r a c t e r i s t i c s that might be expected i n l i g h t of the proposed glycosidase mechanisms. Figure 16: Nojirimycin and Acarbose. Acarbose binds to a-glucosidases up to 10 5 times more t i g h t l y than do the natural s u b s t r a t e s . 1 5 The t i g h t binding observed has been at t r i b u t e d to both the conformation of the hydroxymethylcyclitol r i n g and to i n t e r a c t i o n s between the enzyme c a t a l y t i c groups and the nitrogen atomf 1 The tight binding of nojirimycin could be due to i n t e r a c t i o n s between the enzyme c a t a l y t i c groups and the basic, endocyclic nitrogen and/or by dehydration to a t r a n s i t i o n state analogue as shown, F i g . 16. 1 5 23 Fina l ly , i t is of interest to note that the conformational changes suggested to occur within the enzyme substrate complex during catalysis are not consistent with the 'anti-periplanar lone pair' hypothesis. 1* 2 Recent studies by Sinnott and Hosie 1 4 3 involving the use of 8-deuterium kinetic isotope effects, which are geometry dependent, to probe conformational changes during catalysis by a-glucosidase from yeast, have cast further doubt upon this hypothesis. 24 I.3 Enzyme I s o l a t i o n Before an enzyme may f r u i t f u l l y be studied mechanistically i t i s necessary to pu r i f y i t to homogeneity to ensure that the e f f e c t s observed are due to the enzyme under i n v e s t i g a t i o n , and not to contaminating proteins. Enzymes are b i o l o g i c a l molecules and consequently problems are encountered with t h e i r 'denaturation' or breakdown i f the conditions encountered deviate too far from the p h y s i o l o g i c a l during t h e i r i s o l a t i o n , and the methods employed r e f l e c t t h i s constraint. Enzymes are usually found ei t h e r 'dissolved' i n the cytoplasmic f l u i d within the c e l l or bound to a membrane. The problems associated with the l a t t e r are beyond the scope of t h i s discussion. Once the cytoplasmic f l u i d has been l i b e r a t e d from the c e l l s by the use of such techniques as homogenization, h y d r o l y t i c enzymes (eg. lysozyme), freeze/thaw, u l t r a s o n i c d i s r u p t i o n , osmotic shock, grinding with glass beads or pressure d i f f e r e n t i a l (eg. French pressure c e l l ) , the c e l l debris i s removed by c e n t r i f u g a t i o n . A l l procedures subsequent to t h i s are c a r r i e d out i n a buffer at a pH at which the enzyme i s known to be stable and at between 0-4°C to minimise p r o t e o l y t i c a c t i v i t y . Protease i n h i b i t o r s , such as d i i s o p r o p y l phosphofluoridate, are often added i n order to i n a c t i v a t e p r o t e o l y t i c enzymes. The component of i n t e r e s t i s then i s o l a t e d by a combination of techiques based on differences i n 25 s o l u b i l i t y , s t a b i l i t y , charge, size/shape, a f f i n i t y for a ligand or some other physical property. Nucleic acids are often removed from the c e l l f r e e extract by p r e c i p i t a t i o n with streptomycin sulphate. Following t h i s , d i f f e r e n t i a l p r e c i p i t a t i o n i s almost i n v a r i a b l y c a r r i e d out, usually with ammonium sulphate as p r e c i p i t a n t . A plot of Log. ( s o l u b i l i t y ) vs. io n i c strength for haemoglobin, F i g . 17,^^ shows an i n i t i a l increase i n s o l u b i l i t y due to charge d i s p e r s a l by the s a l t ions. The subsequent decrease i n s o l u b i l i t y has been less well quantified t h e o r e t i c a l l y , but i s thought to be due to a decrease i n available water as water becomes increasingly associated with the hydration spheres of the s a l t i o n s . 4 5 12 0.8 0.4 f 0 ~ -0.8 -12 -1.6 -2.0 Figure 17^: Plot of Log ( s o l u b i l i t y ) vs. Ionic Strength for Haemoglobin. 26 T y p i c a l l y , ammonium sulphate would be added u n t i l the enzyme of int e r e s t was at the point of i n c i p i e n t p r e c i p i t a t i o n ; the contaminating proteins (and polysaccharide) p r e c i p i t a t e d would then be removed by cen t r i f u g a t i o n . More ammonium sulphate would be added u n t i l the protein of i n t e r e s t was just p r e c i p i t a t e d and the supernatant, containing conta-minating protein, discarded. Ammonium sulphate i s the s a l t of choice because of i t s r e l a t i v e inexpense, high s o l u b i l i t y and lack of adverse ef f e c t s on most proteins. Proteins have also been f r a c t i o n a l l y p r e c i p i t a t e d by addition of polyethylene g l y c o l and ethanol, and by adjustment of pH towards the i s o -e l e c t r i c point (the pH at which a protein c a r r i e s no net charge) of the protein of i n t e r e s t . 1 * 7 The l a t t e r method i s of less use as many enzymes are denatured at the i r i s o e l e c t r i c p o i n t s . 4 4 The protein p e l l e t obtained from the above procedure would be dissolved i n a minimum volume of an appropriate buffer (possibly e f f e c t i n g a concentration at th i s stage) and the i o n i c strength lowered by eit h e r d i a l y s i s or gel f i l t r a t i o n . I f the enzyme were unusually stable to extremes of either heat or pH, an extra p u r i f i c a t i o n step, involving p r e c i p i t a t i o n of contaminating proteins by heat or pH treatment, might be inserted at th i s p o i n t . 4 4 > 4 6 Gel f i l t r a t i o n may be used to ef f e c t a further p u r i f i c a t i o n , i n addition to removing s a l t i o n s . 4 7 * 1 + 8 Gel f i l t r a t i o n , or gel exclusion chromatography, i s a chromatographic technique i n which separation on the basis of size and shape takes place. The packing material i s a s o l i d support matrix, based on either a cross-linked polysaccharide (eg. agarose 27 or c e l l u l o s e ) or cross-linked polyacrylamide, which contains pores of such a size as to exclude larger molecules, while f r e e l y admitting smaller ones. Thus, on passing through a column of this material, larger molecules w i l l have less solvent a v a i l a b l e to them than smaller molecules. A f r a c t i o n a t i o n based on si z e (and shape) w i l l take place with the larger molecules being eluted f i r s t . The f r a c t i o n s from the above step containing enzyme a c t i v i t y might then be applied to an ion-exchange column. Ion-exchange chromatography e f f e c t s a separation on the basis of charge. 1 + 7» 1 + 9 Charged groups are covalently attached to a s o l i d support matrix (of a s i m i l a r type to that used for gel f i l t r a t i o n ) and the counter ions are f r e e l y exchangeable with those i n s o l u t i o n . The functional groups commonly used are shown below, Table I I . 4 9 The decision to bind a protein to a cation exchanger (pH below i t s i s o e l e c t r i c point) or an anion exchanger (pH above i t s i s o e l e c t r i c point) depends l a r g e l y on i t s pH s t a b i l i t y range. Once the Impure mixture has been bound to the top of the ion exchange column, unbound proteins are eluted with one or two bed volumes of s t a r t b u f fer. The column i s normally developed with a gradient of increasing i o n i c strength. As the s a l t ions compete for binding s i t e s on the ion exchange matrix, proteins are sequentially desorbed according to the strength of th e i r a f f i n i t y for the ion exchange r e s i n . 28 Table I I 1 * 9 ; Functional Groups used i n Ion-Exchange Materials. Anion Exchangers • 1 Functional Group Aminoethyl (AE-) Diethylaminoethyl (DEAE-) Quaternary aminoethyl (QAE) -OCH 2CH 2NH 3 + -OCH 2CH 2+NH(CH 2CH 3) 2 -OCH^^+NCCjHg) 2CH 2CH(OH)CH 3 -OCH2COO~ -P0 1 +H 2-—CH ^ Cations Exchangers Carboxymethyl (M-) Phospho Sulphopropyl (SP-) The above hypothetical p u r i f i c a t i o n scheme i s representative of some of the more t r a d i t i o n a l techniques commonly used i n protein i s o l a t i o n . Typical p u r i f i c a t i o n and y i e l d s obtained at each step may be seen i n Chapter I I . Other techniques used su c c e s s f u l l y i n protein p u r i f i c a t i o n are: a f f i n i t y techniques (discussed l a t e r ) , hydroxyapatite chromatography (based on C a 2 + and non-specific i n t e r a c t i o n s ) 5 0 , hydrophobic chromato-graphy 5 1 and chromatofocussing 5 2. The l a t t e r technique involves the sett i n g up of a pH gradient across an ion exchange column. Proteins applied to the column migrate, and are focussed, according to the i r r 29 isoelectric points. They are eluted at the pH of these points. Although in principle a very powerful technique, in practice i t is found that many enzymes are denatured at their isoelectric points1*1* lessening practical u t i l i t y . The a f f i n i t y techniques commonly used are af f i n i t y adsorption and aff i n i t y elution chromatography. In the former, an enzyme substrate or inhibitor is covalently attached to a solid support (eg. agarose). The crude protein sample (usually after an i n i t i a l ammonium sulphate p u r i f i -cation step) is applied to the column, the enzyme of interest binds to the immobilised ligand and contaminating proteins are washed off in one bed volume of buffer. The enzyme i s then desorbed and eluted with a pulse of either ligand solution or a (protein) 'deforming' buffer. The application of this technique is illustrated for the case of staphylococcal nuclease,51* Fig. 18. Although this technique has the potential for very large p u r i f i -cations in a single step, this must be weighed against the fact that a packing material, months in the preparation, may be of no use whatsoever. Affinity elution chromatography is a technique in which an Impure protein sample is adsorbed non-specifically to an ion exchange column and the enzyme(s) of interest is eluted with a pulse of enzyme substrate or 30 T 1 r—f f — i r Effiuem (mi) Figure 18: P u r i f i c a t i o n of Stapylococcal Nuclease by A f f i n i t y Adsorption Chromatography. i n h i b i t o r s o l u t i o n . The advantages of th i s technique are the ease with which i t may be attempted ( c f . a f f i n i t y adsorption chromatography) and the large capacity of the mat e r i a l . In a comprehensive study on the p u r i f i c a t i o n of g l y c o l y t i c enzymes 5 5 by a f f i n i t y e l u t i o n i t was pointed out that, i n order to avoid ion exchange e f f e c t s , the desorbing ligand should have either no charge or the same charge as the ion exchange mate r i a l . Due to the non-specific nature of the adsorption this technique i s unsuitable for use with samples i n which the enzyme of i n t e r e s t 31 comprises only a small portion of the t o t a l p r o t e i n b l 3 . The basis of the technique i s either a protein conformational change induced on binding of the desorbing ligand and/or the n e u t r a l i s a t i o n of charge on the protein by charge on the desorbing l i g a n d . 5 5 At least three charges (on the desorbing ligand) had to be introduced per 100,000 Daltons enzyme molecular weight for e l u t i o n of g l y c o l y t i c enzymes. 5 5 For a l l of the above chromatographic techniques d i f f u s i o n i s decreased, and r e s o l u t i o n increased, i f the packing material i s made up of small, monodisperse, sph e r i c a l beads. The use of small bead size requires high pressure to obtain reasonable flow rates and at these (HPLC) pressures the shear forces generated may denature some enzymes. Some of these problems have been reduced by the recent introduction (by Pharmacia) TM of a new packing material (MonoBeads ) for use at medium pressures with TM a, so c a l l e d , Fast Protein L i q u i d Chromatography (FPLC ) system. This material has been u t i l i s e d i n gel f i l t r a t i o n chromatography, ion exchange chromatography and chromatofocussing. The increase i n r e s o l u t i o n that may be obtained by the use of t h i s small, monodisperse packing material i s considerable. A protein sample that eluted as one peak on a column (25 cm x 2.5 cm) of Whatman DE-52 anion exchange r e s i n during a 25 hour run was resolved into fourteen peaks on a MonoBead anion exchange column (5.0 cm x 0.6 cm) i n a 20 minute r u n 5 3 . The decrease i n run time alone, obtained by the use of t h i s material, allows for a much more detai l e d method development. 32 Once an enzyme has been p u r i f i e d by a combination of the above techniques i t i s t y p i c a l l y stored either as a s o l i d suspension c r y s t a l l i s e d with ammonium sulphate, a l y o p h i l i s e d powder, i n s o l u t i o n under toluene vapour or frozen, depending upon i t s s t a b i l i t y to the a v a i l a b l e conditions. Demonstration of protein homogeneity (purity) i s t r a d i t i o n a l l y achieved by observation of any, or a combination, of the following: a single band on denaturing or non-denaturing electrophoresis gels, a single band on an i s o e l e c t r i c focussing gel or the observation of a s i n g l e , symmetrical peak on any chromatographic procedure. Another c r i t e r i o n of purity, for a previously i s o l a t e d enzyme, i s s p e c i f i c a c t i v i t y , i . e . amount of enzyme a c t i v i t y per unit weight of protein. Weight of protein i s determined by a c o l o r i m e t r i c assay (see II.3.1). Electrophoresis i s a method of separation based on charge to mass r a t i o . The support i s usually a porous, cross-linked polyacrylamide gel whose pores are of such a s i z e that the proteins experience a 'sieving' e f f e c t when migrating under the influence of an applied p o t e n t i a l d i f f e r e n c e . The proteins are applied to one end of the gel and migrate under the influence of an applied p o t e n t i a l d i f f e r e n c e . Separation i s thus effected on the basis of both size and charge. If used as a c r i t e -r i o n of homogeneity the procedure should be carried out at a number of pH's to vary the charge to mass r a t i o of the proteins. Proteins denatured with the anionic detergent sodium dodecyl sulphate (SDS) and 33 mercaptoethanol are dissociated into t h e i r component subunits ( i f composed of such) and adsorb a constant weight r a t i o of SDS (1.4 g per g of p r o t e i n ) . 5 6 Electrophoresis of these pretreated proteins i n the presence of SDS thereby e f f e c t s a separation on the basis of molecular weight alone, and i n addition to being a c r i t e r i o n of purity, allows the subunit composition of the protein(s) to be determined. I s o e l e c t r i c focussing i s a method of separation based on i s o -e l e c t r i c point. A pH gradient i s set up across a gel matrix, the protein sample loaded onto the gel and a p o t e n t i a l difference applied. The proteins migrate, and are focussed, to a pH at which they have no net charge, the i s o e l e c t r i c point. Both i s o e l e c t r i c focussing and non-denaturing electrophoresis may be used preparatively, a l b e i t with some d i f f i c u l t y . 34 1.4 Previous Studies on B-Glucosidase from A. f a e c a l i s The f i r s t report of work c a r r i e d out on a B-glucosidase from A. f a e c a l i s was that of i t s p u r i f i c a t i o n and c h a r a c t e r i s a t i o n by Han and S r i n i v a s a n . 5 7 The 8-glucosidase a c t i v i t y was found to be induced by either lactose or c e l l o b i o s e i n the growth media. However, due to the number of mistakes ( a r i t h m e t i c a l , experimental and methodological) found i n this work, i t i s of only l i m i t e d value. Subsequent work on the immobilisation of p a r t i a l l y p u r i f i e d enzyme 5 8 and whole c e l l s 5 9 onto s o l i d supports has been published. Work published as a Ph.D. thesis by E.O. Smith under the supervision of S r i n i v a s a n 6 0 suggested that a number of carbohydrates are a c t i v e l y transported across the c e l l wall membrane of A. f a e c a l i s . Gel chromatography of c e l l free extracts yielded two active f r a c t i o n s with d i f f e r e n t B-glucosidase to B-galactosidase a c t i v i t y r a t i o s . The B-glucosidase peak was p u r i f i e d to apparent homogeneity by preparative d i s c - g e l electrophoresis. The s p e c i f i c a c t i v i t y of the pure protein was 55 units/mg; one unit of enzyme i s that amount necessary to hydrolyse one umole of p-nitrophenyl-B-D-glucopyranoside i n one minute at pH 6.5 i n 0.1M sodium phosphate buffer at 40°C. A comparison of these r e s u l t s with those found herein w i l l be made i n Chapter I I I . CHAPTER II Experimental 36 Abbreviations Used PMR proton magnetic resonance TLC t h i n layer chromatography SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis FPLC fast protein l i q u i d chromatography IR i n f r a - r e d spectroscopy UV u l t r a - v i o l e t TMS tetramethylsilane DSS 2,2-dimethyl-2-silapentane-5-sulphonic a c i d , sodium s a l t hydrate PNP p-nitrophenol PMSF phenylmethylsulphonyl f l u o r i d e EDTA ethylaminediamenetetraacetic acid MOPS 3-(N-morpholino)-propanesulphonic acid SDS sodium dodecyl sulphate PNP G p-ni t r opheny1-6-D-gluco pyr ano s ide PNPGal p-nitrophenyl-6-D-galactopyranoside PNPMan p-nitrophenyl-6-D-mannopyranoside PY-G 6-D-glucopyranosyl pyridinium bromide 2,4-DNPG or DNPG 2,4-dinitrophenyl-6-D-glucopyranoside 2,4-TDNPG or TDNPG 2,4-dinitrophenyl-8-D-l-thioglucopyranoside BSA bovine serum albumin DNA deoxyribonucleic acid RNA ribonucleic acid DNAse deoxyribonuclease 2,4-DNP dinitrophenol 2,4-TDNP thiodinitrophenol ppm parts per m i l l i o n A l l e x t i n c t i o n c o e f f i c i e n t s are i n units of: 1 m o l - 1 cm"1 37 II.1 Synthesis II.1.1 General Methods Infra-red spectra were recorded on a Nicolet 5D-X Fourier trans-form instrument, samples being prepared as a nujol mull held between sodium chloride p l a t e s . FT proton magnetic resonance (PMR) spectra were recorded either on a Bruker WP 80, a Bruker WH 400 or an instrument consisting of a 270 MHz Oxford superconducting magnet, a Nicolet 1180 computer, a Nicolet 293b pulse programmer and a Bruker WH 60 console. Deuterated solvents were used with either TMS or DSS standard. C 1 3{ 1H} magnetic resonance (CMR) spectra were recorded on a Bruker WH 400 at 100.6 MHz with TMS as standard. Melting points were determined on a Kofler micro heating stage and are uncorrected. Evaporations at reduced pressure were c a r r i e d out i n a BUchi rotary evaporator at reduced pressure under water pump vacuum at the optimum temperature for the removal of solvent without decomposition of the material being prepared. P u r i f i c a t i o n by f l a s h chromatography was car r i e d out according to the method of Clark et a l 7 4 i n the solvent s p e c i f i e d . Thin layer chromatography (TLC) was carried out on aluminum backed K i e s e l g e l 60 ^ 25^ plates supplied by Merck Chemical Company and v i s u a l i s e d by u l t r a - v i o l e t absorbance (UV) and/or charring by spraying with 10% HjSO^ 38 i n methanol and heating at 100°C for 5 minutes (H 2S0 4). Solvent A consists of ethyl acetate:ethanol:water 7:2:1 and i s most useful for the separation of free sugars. Solvent B consists of pentane:ethyl acetate:-ethanol 20:9:1 and i s most useful for the separation of acetylated sugars. Solvent C consists of chlor o f o r m : a c e t o n i t r i l e : e t h y l acetate 3:1:1 and i s an aprotic solvent suitable for acetylated sugars. Solvent D consists of methyl ethyl ketone:petroleum ether 30-60 1:3. Solvents used were p u r i f i e d as described below: Acetone was dried by d i s t i l l a t i o n from 4A molecular sieves and stored over 4A molecular sieves under dry nitrogen. Methanol was dried by d i s t i l l a t i o n from magnesium methoxide and stored over 3A molecular sieves under dry nitrogen. Pyridine was dried over potassium hydroxide, d i s t i l l e d from barium oxide and stored over potassium hydroxide under dry nitrogen. Dimethyl formamide was dried by d i s t i l l a t i o n at reduced pressure from calcium hydride and stored over 4A molecular sieves under dry nitrogen. A c e t o n i t r i l e was dried by d i s t i l l a t i o n from 4A molecular sieves and stored over 4A molecular sieves under dry nitrogen. P-Nitrophenol was p u r i f i e d by repeated r e c r y s t a l l i s a t i o n from 0.01M hydrochloric a c i d . MP = 115°C. Nitrogen was dried by passage through a column of calcium c h l o r i d e . A l l chemicals used were general purpose grade reagents unless otherwise s p e c i f i e d . A l l products were dried for at least 24 hours In a vacuum dessicator over phosphorus pentoxide. 39 I I . 1.2 p-Nitrophenyl - B-D-cellobioside, IV The t i t l e compound was prepared from cellobiose by the following method. 1,2,3,6,2',3*,4',6'-Octa-0-acetyl-q-D-cellobiose, I, was prepared by the method of Wolfrom and Thompson 6 2 with the following modifications. Cellobiose (10 g, 0.029 Mol.) was added to a s t i r r e d , cooled (0°C) mixture of acetic anhydride (70 cm3, 0.74 Mol.) and pyridine (100 cm 3). After 72 hours the reaction had not gone to completion (TLC, solvent A) and undissolved s o l i d remained i n the mixture. A further 25 cm3 of pyridine was added to e f f e c t d i s s o l u t i o n . After 100 hours the reaction was worked up as d e s c r i b e d 6 2 . Y i e l d = 17.7 g, 90%. Melting point = 198-200°C. L i t . Melting p o i n t 6 3 = 202-202.5°C. Rf (Solvent A) = 0.75 (H 2SO l 4). 2,3,6,2',3',4' )6'-hepta-O-acetyl-a-D-cellobiosyl bromide (acetobromocellobiose), I I , was prepared according to the method of Stanek and Kocourek 6 4 by reaction of I with hydrogen bromide i n chloroform and a c e t i c a c i d . Y i e l d = 64%. Melting point = 186-188°C. L i t . Melting p o i n t 6 4 = 188°C. Rf (solvent C) = 0.60 (H 2SO l +). p-Nitrophenyl 2,3,6,2',3',4',6'-hepta-O-acetyl -B-D-cellobioside, I I I , was prepared by the method of Capon 8 7 with the following modifications. 11(1.0 g, 0.0015 Mol.) and p-nitrophenol (0.41 g, 0.0029 40 Mol.) were dissolved i n dry acetone (12 cm 3). Potassium carbonate (0.86 g, 0.0062 Mol.) was added and the mixture refluxed for 17 hours at which time TLC (solvent B) showed a major spot v i s u a l i s e d by both UV and H 2S0^. The reaction was worked up as d e s c r i b e d 8 7 . Y i e l d = 0.30 g, 28%. Melting point = 238-240°C. L i t . Melting p o i n t 6 5 = 235°C. Rf (solvent C) = 0.54 (UV, H 2S0 4). p-Nitrophenyl-8-D-cellobioside, IV, was prepared by c a t a l y t i c deacetylation of III using sodium methoxide i n methanol according to Zemplen 6 6. The gum obtained was t r i t u r a t e d with d i e t h y l ether to Induce c r y s t a l l i s a t i o n and r e c r y s t a l l i s e d from water. Y i e l d = 94%.. Melting point = 255-256.5°C. L i t . Melting p o i n t 6 5 = 255-256°C. Rf (solvent A) = 0.43 (UV, H^O^). Elemental analysis c a l c d . for C 1 8H 2 5N0 1 3.2H 20: C, 43.30%; H, 5.85%; N, 2.81%; 0, 48.04%. Found: C, 43.09%; H, 5.95%; N, 2.78%; 0, 48.10%. PMR (80 MHz, D 20): 7.28, 8.30 (2 x dd; J = 9.0, 2.0 Hz; 2H each; aromatic H's), 5.35 (d; J = 6.2 Hz; 1H; H - l ) , 4.59 (d; J = 8.4 Hz; 1H; H-7), 3.3-4.2 (m; 12H; H-2-H-6 and H-2'-H-6'). II.1.3 p-Nitrophenyl-B-D-glucopyranoside, VIII The t i t l e compound was prepared from glucose by the following method. 1,2,3,4,6-Penta-0-acetyl-a-D-glucopyranose, V, was prepared according to the method of Wolfrom and Thompson 6 2 by reaction of glucose 41 with acetic anhydride i n the presence of perc h l o r i c a c i d . Y i e l d = 55%. Melting point = 111.5-112.5°C. L i t . Melting p o i n t 6 2 = 112-113°C. 2,3,4,6-Tetra-O-acetyl-g-D-glucopyranosyl bromide (acetobromoglucose), VI, was prepared from glucose i n a 'one pot' reaction according to the method of Lemieux 6 7. Glucose was added to an acetyl a t i n g mixture consisting of ac e t i c anhydride and perchloric acid and subsequent addition of bromine and red phosphorus (to produce phosphorus tribromide i n s i t u ) converted the pentaacetate, V, to the bromide, VI. An alternate procedure u s e d 6 8 was conversion of the c r y s t a l l i n e pentaacetate, V, to the bromide by reaction with hydrogen bromide i n ace t i c acid and chloroform ( c f . preparation of I I ) . The material was stored at -20°C. For both preparations: Y i e l d > 80%. Melting point = 88°C. L i t . Melting p o i n t 6 7 = 88-89°C. Rf (solvent C) = 0.55 (HjSO^). p-Nitrophenyl-2,3,4,6-tetra-O-acetyl -B-D-glucopyranoside, VII, was prepared by the method of Capon 8 7 with the following modifications. The bromide, VI, (15 g, 0.062 Mol.) and p-nitrophenol (10 g, 0.072 Mol.) were dissolved i n dry acetone (150 cm3) and potassium carbonate (12 g, 0.086 Mol,) added. The s t i r r e d mixture was refluxed for 4 hours. TLC (solvent D) showed 2 major spots, Rf = 0.26 (UV, H 2S0 4) and 0.20 (H 2S0 k). The reaction was worked up as d e s c r i b e d 8 7 and r e c r y s t a l l i z a t i o n of the product from methanol yielded the UV active material e x c l u s i v e l y . 42 Y i e l d = 4.0 g, 21%. Melting point = 174.5-175°C. L i t . Melting p o i n t 6 8 = 174-175°C. Rf (solvent D) = 0.26 (UV, HjSO^). Cooling of the mother l i q u o r induced c r y s t a l l i s a t i o n of the impurity which was i d e n t i f i e d by PMR (270 MHz) as the product of an elimination reaction, 2-acetoxy-3,4,6-tri-0-acetyl-D-glucal. PMR (270 MHz, CDC13): 2.13 (m; 12H; CH3CO-), 4.28 (dd; J = 10, 3.3 Hz; 1H; H-5), 4.46 (m; 2H; H-6), 5.26 ( t ; J = 4.0 Hz; 1H; H-4), 5.61 (d; J = 4.0 Hz; 1H; H-3), 6.72 (s; 1H; H - l ) . Carrying out the reaction at room temperature did not increase the y i e l d of the desired product, VII. p-Nitrophenyl - B-D-glucopyranoside, VIII, was prepared by c a t a l y t i c deacetylation of VII using sodium methoxide i n methanol according to the method of Zemplen 6 6. Y i e l d = 70%. Melting point = 164.5-165.5°C. L i t . Melting p o i n t 6 8 = 164°C. Rf (solvent A) = 0.63 (UV, HjSO^). PMR (80 MHz) i d e n t i c a l to that of authentic sample. II.1.4 Isopropyl - B-D-l-thioglucopyranoside, XII The t i t l e compound was prepared from the bromide, VI, by the following method. 2-(2,3,4,6-Tetra-0-acetyl -B-D-glucopyranosyl)-2-thiopseudourea hydrobromide, IX, was prepared by reaction of the bromide, VI, with thiourea i n dry acetone according to the method of Cerny et a l 6 9 . Y i e l d = 78%. Melting point = 195°C. L i t . Melting p o i n t 7 0 = 205°C. PMR (270 MHz, D 20): 2.10 (m; 12H; CH 3C0-), 4.12-4.60 (m; 3H; H-5, H-6), 5.26 43 ( t ; J = 8.5 Hz; 1H; H-=2), 5.38 (d, J = 8.5 Hz; 1H; H - l ) , 5.82 ( t ; J = 8.5 Hz; 2H; H-3, H-4). Repeated attempts to prepare 2,3,4,6-tetra-0-acetyl-l-thio-8-D- glucopyranoside, X, by addition of IX to a saturated s o l u t i o n of potassium carbonate i n water according to the method of Cerny et a l 6 9 f a i l e d . Addition of carbon t e t r a c h l o r i d e to extract the product as i t was formed according to the method of Cerny and Pacak 7 1 gave X i n good y i e l d . Y i e l d = 83%. Melting point = 115.5-116.5°C. L i t . Melting p o i n t 7 0 = 115°C. Rf (solvent B) = 0.40 (HjSO^). PMR (270 MHz, CDC13): 2.05 (m; 12H; CH3CO-), 2.34 (d; J = 10 Hz; 1H; SH), 3.75 (m; 1H; H-5), 4.13 (dd; J = 12, 2.8 Hz; 1H; H-6), 4.26 (dd; J = 12, 4.0 Hz; 1H; H-6), 4.56 ( t ; J = 10Hz; 1H; H - l ) , 5.00, 5.11, 5.22 (3 x t; J = 8.5 Hz; 2H each; H-2, H-3, H-4). On D 20 shake resonance at 2.34 ppm disappeared and that at 4.56 ppm collapsed to a doublet. 2,3,4,6-Tetra-O-acetyl isopropyl - B-D-l-thioglucopyranoside, XI, was prepared by the reaction of X with isopropyl iodide and potassium carbonate i n dry acetone according to the method of Cerny and Pacak 7 2. The discrepancy i n the melting points can be ascribed to d i f f e r e n t c r y s t a l forms as the PMR spectrum i s i n accord with the structure. Y i e l d = 82%. Melting point = 108.5-109°C. L i t . Melting p o i n t 7 3 = 91°C. Rf ( d i e t h y l ether) = 0.62 (H 2 S0 H ) . Proton magnetic resonance (80 MHz, CDCI3): 1.35 (d; J = 7.0 Hz; 6H; isopropyl CH 3), 2.06 (m; 12H; CH 3C0-), 3.21 (septuplet; J = 7 Hz); 1H; isopropyl CH), 3.75 (m; 1H; H-5), 4.23 (m; 2H; H-6), 4.63 (d; J = 10 Hz; 1H; H - l ) , 4.88-5.50 (m; 3H; H-2, H-3, H-4). 44 Isopropyl-B-D-l-thioglucopyranoside, XII, was prepared by c a t a l y t i c deacetylation of XI using sodium methoxide i n methanol according to the method of Zemplen 6 6. The material was p u r i f i e d by f l a s h chromatography 7 4 i n methanol:chloroform 3:7. The hygroscopic gum obtained c r y s t a l l i s e d on standing at -20°C for 3 months. Y i e l d = 91%. Elemental analysis c a l c d . for (gum) C 9H 1 80 5S: C, 45.37%; H, 7.61%; 0, 33.56%. Found: C, 44.79%; H, 7.62%; 0, 33.72%. PMR (400 MHz, D 20): 1.35 (dd; J = 8.0, 2.0 Hz; 6H; isopropyl CH 3), 3.27-3.62 (m; 6H; SH, H-2, H-3, H-4, H-5, isopropyl CH), 3.74 (dd; J = 14, 5.6 Hz; 1H; H-6), 3.94 (dd, J = 14, 2.4 Hz; 1H; H-6): 4.65 (d; J = 9.2 Hz; 1H; H - l ) . II.1.5 g-D-Glucopyranosyl Pyridinium Bromide, XIV The t i t l e compound was prepared from the bromide, VI, by the following method. 2,3,4,6-Tetra-O-acetyl-B-D-glucopyranosyl pyridinium bromide, XIII, was prepared from the bromide, VI, by reaction with dry pyridine i n the presence of m-cresol according to the method of Sinnott and W i t h e r s 3 4 . Y i e l d = 25%. Melting point = 168-169°C. L i t . Melting p o i n t 7 5 = 170°C. PMR (80 MHz, D 20) i d e n t i c a l to that r e p o r t e d 7 5 . B-D-Glucopyranosyl pyridinium bromide, XIV, was prepared by deacetylation of XIII with methanolic hydrogen bromide according to the method of Lemieux and Morgan 7 5. Y i e l d = 80%. Melting point = 176°C. L i t . Melting p o i n t 7 5 = 176-177°C. 45 PMR (80 MHz, D 20) i d e n t i c a l to that r e p o r t e d 7 5 . I I . 1.6. B-D-Glucopyranosyl Azide, XVI The t i t l e compound was prepared from the bromide, VI, by the following method. 2,3,4,6-Tetra-0-acetyl -B-D-glucopyranosyl azide, XV, was prepared from the bromide, VI, by reaction with sodium azide i n dry a c e t o n i t r i l e according to the method of Szarek et a l 7 6 . Y i e l d = 72%. Melting point = 127-128°C. L i t . Melting p o i n t 7 6 = 127.5-129°C. Rf (solvent B) = 0.30 (UV, H 2S0 4). PMR (80 MHz, CDC13) i d e n t i c a l to that r e p o r t e d 7 6 . IR i d e n t i c a l to that r e p o r t e d 7 6 . B-D-Glucopyranosyl azide, XVI, was prepared by c a t a l y t i c deacetylation of XV using sodium methoxide i n methanol according to the method of Zemplen 6 6. Y i e l d = 81%. Melting point = 94°C. L i t . Melting p o i n t 7 6 = 89°C. 1 3 C magnetic resonance (acetone-d 6) i d e n t i c a l to that r e p o r t e d 7 6 . IR i d e n t i c a l to that reported. Elemental analysis c a l c d . for CgH^OgNg: C, 35.12%; H, 5.40%; N, 20.48%. Found: C, 35.11%; H, 5.36%, N, 20.33%. I I . 1.7 B-D-Glycopyranosyl Fluoride, XVIII The t i t l e compound was prepared from the bromide, VI, by the following method. 2,3,4,6-Tetra-0-acetyl-8-D-glucopyranosyl f l u o r i d e , XVII, was prepared from the bromide, VI, by reaction with s i l v e r f l u o r i d e (AgF) i n 46 dry a c e t o n i t r i l e according to the method of H e l f e r i c h ' ' . Y i e l d = 65%. Melting point = 86°C. L i t . Melting point = 98°C ( a f t e r repeated r e c r y s t a l l i s a t i o n ) . 8-D-Glucopyranosyl f l u o r i d e , XVIII, was prepared by c a t a l y t i c deacetylation of XVII using sodium methoxide i n methanol according to the method of Zemplen 6 6 with the following modifications. The reaction was only allowed to proceed for 5 minutes before working up. The material had to be p u r i f i e d by f l a s h chromatography 7 1 4 i n solvent A. An alternate procedure involved carrying out the deacetylation at 0°C. In t h i s case c r y s t a l l i s a t i o n could be induced without further p u r i f i c a t i o n . However, thi s l a t t e r material i n a c t i v a t e d the enzyme, presumably because i t contained trace amounts of s i l v e r . Y i e l d = 27%. Melting point = 90°C. L i t . Melting p o i n t 7 8 = 99-102°C (aft e r extensive r e c r y s t a l l i s a t i o n ) . Rf (solvent A) = 0.40 (H 2S0 1 +). Elemental analysis c a l c d . for C 6 H u 0 5 F : C, 39.56%; H, 6.08%. Found: C, 39.48%; H, 6.04%. PMR (270 MHz, D 20) 3.35-3.60 (m;4H; H-2, H-3, H-4, H-5), 3.75 (dd; J = 14, 4.4 Hz; 1H; H-6), 3.90 (dd, J = 14, 1.2 Hz; 1H; H-6), 5.22 (dd, J = 53, 7.6 Hz; 1H; H - l ) . II.1.8 B-D-Glucopyranosylamine, XIX 8-D-Glucopyranosylamine, XIX, was prepared from glucose by reaction with methanolic ammonia according to the method of Cusack et a l 7 9 . Y i e l d = 91%. Melting point = 126-129°C. L i t . Melting p o i n t 8 0 = 47 126-128°C. II.1.9 2,4-Dinitrophenyl-l-thio-8-D-glucopyranoside, XXI The t i t l e compound was prepared from the acetylated thio sugar, X, as follows. 2,3,4,6-Tetra-0-acetyl-l-(2,4-dinitrophenyl)-l-thio-B-D- glucopyranoside, XX, was prepared as follows. X (4g, 0.010 mol.), 2,4-dinitro-l-fluorobenzene (1.92 g, 0.010 mol.) and 1,4-diazabicyclo-[2,2,2]octane (0.45 g, 0.0040 mol.) were dissolved i n dry dimethyl formamide (30 cm 3). The sol u t i o n r a p i d l y went red and a f t e r 100 minutes TLC (solvent B) indicated that the reaction had gone to completion. The reaction mixture was evaporated to a gum under reduced pressure, dissolved i n chloroform (250 cm3) and the solution washed with successive 100 cm3 portions of saturated sodium bicarbonate solution and water. This so l u t i o n was then dried over anhydrous magnesium sulphate, decolourised with charcoal and evaporated under reduced pressure to y i e l d a f a i n t l y yellow gum. C r y s t a l l i s a t i o n and r e c r y s t a l l i s a t i o n were c a r r i e d out by p r e c i p i t a t i o n from chloroform with low b o i l i n g petroleum ether. Y i e l d = 5.6 g; 59%. Melting point = 197°C. L i t . Melting p o i n t 8 1 = 200-201°C. Rf (solvent B) = 0.45 (UV, HgSO^). PMR (270 MHz, CDCI3) 2.06 (m; 12H; CH3CO), 3.96 (m; 1H; H-5), 4.22 (dd; J - 12, 2.8 Hz; 1H; H-6), 4.28 (dd; J = 12, 4.4 Hz; 1H; H-6), 5.00 (d; J = 9.6 Hz; 1H; H- l ) , 5.17, 5.21, 5.34 ( 3xt; J - 8.4 Hz each; 1H each; H-2, H-3, H-4), 7.98 (d; J = 8.8 Hz; 1H; H-6' of phenyl), 8.42 (dd; J = 8.8, 2.0 Hz; 1H; H-5' of 48 phenyl), 9.02 (d; J = 2.0 Hz; 1H; H-3' of phenyl). 2,4-Dinitrophenyl - B-D-l-thioglucopyranoside, XXI, was prepared by c a t a l y t i c deacetylation of XX using sodium methoxide i n methanol according to the method of Zemplen 6 6. The f i n a l product was found to have one acetone of c r y s t a l l i s a t i o n associated with i t . A peak i n the proton magnetic resonance spectrum at 2.10 ppm, integrating to 3 protons, disappeared i f the sample was evaporated to a gum and redissolved. The Elemental analysis i s consistent with one acetone of c r y s t a l l i s a t i o n . Y i e l d = 74%. Melting point (from methanol/acetone) = 111°C. L i t . Melting point (from methanol/diethyl e t h e r ) 8 1 = 184-185°C. Elemental analysis c a l c d . for C 1 2H 1 40 9N 2S:CH 3COCH 3: C, 42.85%; H, 4.79%; N, 6.66%; S, 7.64%. Found: C. 42.59%; H, 4.72%; N, 6.72%; S, 7.79%. PMR (400 MHz, DMS0-d6) 3.20-3.55 (m; 5H; H-2, H-3, H-4, H-5, H-6), 3.75 (dd; J = 12, 4.4 Hz; 1H; H-6), 4.55, 5.08, 5.23, 5.67 (4xs; 1H each; 2-OH, 3-OH, 4-OH, 6-0H), 5.00 (d; J = 7.6 Hz; 1H; H - l ) , 8.05 (d; J = 8.0 Hz; 1H; H-6' of phenyl), 8.40 (dd; J = 8.0, 1.6 Hz; 1H; H-5' of phenyl), 8.85 (d; J = 1.6 Hz; 1H; H-3' of phenyl). On D 20 shake resonances at 4.55, 5.08, 5.23 and 5.67 ppm disappeared. 49 II.2 Growth of A. f a e c a l i s , ATCC 21400, and Preparation of C e l l Free  Extract II.2.1 General Methods pH was measured on a Radiometer PHM-82 d i g i t a l pH meter equipped with a Sigma Trizma glass/calomel combination electrode and standardised with Radiometer standard b u f f e r s . A l l measurements of o p t i c a l density were c a r r i e d out on a Pye-Unicam PU-8800 u l t r a - v i o l e t / v i s i b l e recording spectrophotometer i n 1 cm path length glass or quartz c e l l s . Centrifugation was c a r r i e d out on a S o r v a l l RC-5B re f r i g e r a t e d centrifuge equipped with either a SS-34 (350 cm3 capacity) or a GS-3 (2.5 l i t e r capacity) rotor at 4°C. A l l media were s t e r i l i s e d at 121°C either i n an autoclave (American S t e r i l i z e r Co. Model AS-DIT616GE) or d i r e c t l y i n the 20 l i t r e fermenter, which i s steam jacketed. Small volumes of media were auto-claved at 121°C for 20 minutes and larger volumes (greater than 10 l i t r e s ) for 45 minutes. The component solutions were s t e r i l i s e d separately as shown below (II.2.2). The French pressure c e l l used was an American S c i e n t i f i c Bragg 6600. pH 6.8 buffer r e f e r s to 100 mM sodium phosphate buffer. This was prepared by mixing appropriate amounts of di-sodium hydrogen ortho-phosphate (A.R.) and sodium di-hydrogen orthophosphate, making up to 50 volume i n double deionised water and then further adjusting the pH with 1M sodium hydroxide or 1M hydrochloric acid as necessary. The t u r b i d i t y of the c e l l cultures was quantified by measuring the absorbance at 660 nm. B-Glucosidase a c t i v i t y was quantified by adding between 10 and 100 uL (as necessary) of the sample to be assayed to an 1 cm pathlength cuvette containing 3.00 cm3 of a so l u t i o n of p-nitrophenyl-8-D-gluco-pyranoside (PNPG) (greater than 5.2 mM, l e . 12 Km) i n pH 6.8 b u f f e r . A l l weighings were ca r r i e d out on either micro or semi-micro balances and a l l accurate volumes were measured with either grade A a n a l y t i c a l glassware or Hamilton M i c r o l i t r e syringes. The cuvette was e q u i l i b r a t e d to 37.0°C (±0.1°C) i n the spectrophotometer, by means of a Julabo VI c i r c u l a t i n g thermostat bath, p r i o r to addition of the sample to be assayed. Optical density readings at a wavelength of 400 nm were taken every 100 s for up to 1000 s. Total units of 6-glucosidase a c t i v i t y i n the o r i g i n a l sample were calculated from the following equation: U = xy(0Dc)(0.6xlQ 6) = 0.0834xy(0Dc) 7.28zxl0 ; ixl0 ; 1 z where: U i s the number of units B-glucosidase a c t i v i t y ; 1 unit being defined as that amount of 6-glucosidase which hydrolyses 1 umole of p-nitrophenyl - B-D-glucopyranoside (PNPG) i n 1 minute at pH 6.8 and 37°C; ODc i s the average change i n absorbance observed at 400 nM i n 100 s; 51 x i s the t o t a l sample volume; y i s the volume i n the cuvette; z i s the sample volume added to the cuvette and 7.28 x 10 3 i s the e x t i n c t i o n c o e f f i c i e n t of p-nitrophenol at pH 6.8 and 37°C. Media nutrients were obtained from Difco Laboratories. Lactose was either BDH b a c t e r i o l o g i c a l grade or general purpose reagent grade. Deoxyribonuclease (DNAse) was deoxyribonuclease I from bovine pancreas containing 10,000 Dornase units/mg, obtained from Calbiochem-Behring, #260912. p-Nitrophenyl-8-D-glucopyranoside (PNPG) was either prepared as described (II.1.3) or obtained from the Sigma Chemical Company. Phenylmethylsulphonyl f l u o r i d e (PMSF) and the stock b a c t e r i a l cultures were supplied by W. Wakarchuk, Microbiology department, UBC. A l l other materials were general purpose reagent grade unless otherwise stated. II.2.2 Growth of A. f a e c a l i s A. f a e c a l i s stock culture was supplied as either a stab culture i n soft agar containing Difco nutrient (stored at 4°C) or as a 50% culture suspension i n g l y c e r o l (stored at -20°C). These preparations were used to innoculate 100 cm3 cultures which were themselves used either for tests on a small scale or as innoculum for 10-20 l i t r e c u l t ures. Two media were used: 52 1) Minimal Salts Medium 5 7 NaCl 0.30% (NH^SO^ 0.10%/ KH2P01+ 0.05% K2HPOi| 0.05% MgSO^ 0.01% C a C l 2 0.01% Difco Yeast extract 0.50%' Lactose 3.00%' Luria B r o t h 8 2 Difco Tryptone 1.00% Difco Yeast extract 0.50% NaCl 0.05% Lactose 3.00%' •Autoclaved separately ^Autoclaved separately pH adjusted to 7.0 with 1.0M sodium hydroxide. N.B. a) 0.1% ce l l o b i o s e could be used i n place of lactose to induce B-glucosidase p r o d u c t i o n 8 2 . b) If yeast extract was omitted from the minimal s a l t s medium (preparation as g i v e n 5 7 ) , the c e l l y i e l d was about 10% of that obtained when i t was included. c) Both successful p-glucosidase i s o l a t i o n s were c a r r i e d out from material cultured i n Lur i a broth although t h i s medium gave s l i g h t l y less t o t a l p-glucosidase a c t i v i t y (as measured i n the crude culture) and a s l i g h t l y lower c e l l y i e l d . 53 100 cm3 Cultures were grown i n 500 cm3 Erhlenmeyer f l a s k s on a thermostatted platform shaker with a 7/8" throw ( b u i l t by the mechanical shop, Chemistry Department, U.B.C.) at 30°C and 200 rpm. 10-20 l i t r e cultures were grown i n impeller driven, steam-jacketed 20 l i t r e fermenters ( b u i l t by the mechanical shop, Chemistry Department, U.B.C.) at 30°C, 500 rpm and aerated at 500 cm3 per l i t r e . Polypropylene g l y c o l (2-5 cm3) was added to the 10-20 l i t r e cultures i n order to minimize foaming. B-Glucosidase a c t i v i t y could be monitored i n whole c e l l s i n the culture medium. The v a r i a t i o n of c e l l density and 8-glucosidase a c t i v i t y with time, for a 13 l i t r e culture grown i n minimal s a l t s medium, innocu-lated with 150 cm3 of l a t e log phase culture, i s shown below, Table I I I , F i g . 19. The three phases of b a c t e r i a l growth: lag phase, log phase and stationary phase, are also marked, F i g . 19. I t should be noted that the c e l l y i e l d (and concomitant B-glucosidase a c t i v i t y ) i s somewhat lower i n the 100 cm3 cultures (OD 660~4) than i n the 10-20 l i t r e cultures (OD 660~6-8). This i s due to the forced aeration i n the 10-20 l i t r e c u l t u r e s . C l e a r l y i t i s important that the cultures be harvested at the lat e log growth phase for maximum 8-glucosidase y i e l d . The decrease i n B-glucosidase a c t i v i t y observed (about 20% over 3 hours) a f t e r the stationary phase had been reached suggests that the organism a c t i v e l y metabolises the enzyme. C e l l free extracts, i n the absence of protease i n h i b i t o r s , show only a 25% decrease i n a c t i v i t y over 60 hours. 54 Table I I I ; V a r i a t i o n of C e l l Density and B-Glucosidase A c t i v i t y With Time. Time (hours) OD 660 Total A c t i v i t y (p-glucosidase i n 10 L units) S p e c i f i c A c t i v i t y (units/OD 660) 0.0 0.00 0 0 3.5 0.34 0 0 5.5 1.06 82 77 11.5 4.83 1414 292 12.5 5.32 1643 310 14.0 5.88 1847 311 16.5 5.92 1480 251 E 0 5 10 15 20 25 30 Time (hours) Figure 19. V a r i a t i o n of C e l l Density, S p e c i f i c A c t i v i t y and B-Glucosidase A c t i v i t y with Time. 55 When cent r i f u g a t i o n was c a r r i e d out 4-5 hours after the stationary phase had been reached, 50-70% of the B-glucosidase a c t i v i t y was found to be associated with the supernatant. This suggests that c e l l a u t o l y s i s had started to occur. During the growth of one 10 l i t r e culture aeration f a i l e d between the early and mid-log phases. The noxious smell, together with the reasonably high c e l l y i e l d and p-glucosidase a c t i v i t y found i n the mature culture, suggest that the organism i s a f a c u l t a t i v e anaerobe. Once a 10-20 l i t r e culture had reached l a t e log/early stationary growth phase i t was transferred to the cold room (4°C) and the c e l l s harvested immediately by c e n t r i f u g a t i o n i n 2.5 l i t r e batches on GS-3 rotor at 5000 rpm for 20 minutes. The c e l l p e l l e t was stored i n the freezer at -20°C u n t i l i t was required. II.2.3 p-Glucosidase Induction Studies It i s known 8 3 that isopropyl - p-D-thiogalactose induces the synthesis of p-galactosidase i n E. c o l i . This compound i s not hydrolysed by p-galactosidase and therefore only a small amount has to be added to the culture i n order to induce i t s production. It was thought that i s o p r o p y l - p - D - l - t h i o g l u c o s e , XII, might induce synthesis of p-glucosidase i n A. f a e c a l i s and this was investigated. A minimal s a l t s medium, containing 0.5% sodium acetate as a carbon source i n place of lactose, was prepared and the pH adjusted to 7 with g l a c i a l a c e tic a c i d . 100 cm3 of t h i s medium was innoculated with 56 A. f a e c a l i s , incubated at 30°C on a platform shaker and the culture allowed to reach early stationary phase. The c e l l y i e l d was about 20% that of a control culture grown i n Luria broth and no 8-glucosidase a c t i v i t y could be detected. Five units of a c t i v i t y were found i n the control culture. The experiment was repeated and when the acetate culture had reached early log phase (at the f i r s t sign of v i s i b l e t u r b i d i t y ) 0.05% isopropyl - 8-D-l-thioglucose was added. No 8-glucosidase a c t i v i t y was detected at this or at any subsequent growth stage of t h i s c u l t u r e . B-Glucosidase synthesis i s probably not, therefore, induced i n A. f a e c a l i s by the thioglucose d e r i v a t i v e , XII. II.2.4 Preparation of C e l l Free Extract From A. f a e c a l i s ; C e l l Breakage Four methods of c e l l l y s i s were investigated; lysozyme and freeze/thaw treatment, French pressure c e l l , sonication, and b a l l i s t i c d i s i n t e g r a t i o n with glass beads. 1) Lysozyme and Freeze/Thaw Treatment 8 1 4 This method of c e l l breakage r e l i e s upon the hydrolysis of the c e l l wall polysaccharide by hen eggwhite lysozyme and disruption of the c e l l wall by repeated cycles of freezing and thawing. Two 100 cm3 cultures grown in minimal s a l t s medium and allowed to reach l a t e log growth phase contained a t o t a l of 10 units of B-glucosidase a c t i v i t y . After c e n t r i f u g a t i o n for 10 minutes at 9000 rpm i n a GS-3 rotor, no p-glucosidase a c t i v i t y was detected i n the supernatant. The c e l l p e l l e t (3.6 g wet weight) was s p l i t into two equal portions. One 57 portion was resuspended i n 20 cm^ of pH 6.8 b u f f e r . To this was added 20 mg of hen eggwhite lysozyme, 10 uL °f a 250 mM EDTA so l u t i o n , and 0.1 mg DNAse. This mixture was incubated at 30°C for 30 minutes, r a p i d l y frozen ( i n a dry ice/acetone bath) and thawed at 30°C. The process was repeated twice more. After c e n t r i f u g a t i o n for 15 minutes at 15,000 rpm i n a SS-34 rotor, the supernatant and p e l l e t (resuspended i n 20 cm3 of pH 6.8 buffer) were both assayed. The t o t a l a c t i v i t y recovered (supernatant and p e l l e t ) was 67% of the o r i g i n a l a c t i v i t y . The a c t i v i t y associated with the supernatant was 23% of the recovered a c t i v i t y . C e l l breakage was poor. If t h i s procedure was c a r r i e d out on c e l l s i n which autolysis had begun, c e l l breakage was greater than 90%. 2) French Pressure C e l l 8 1 * In t h i s method of c e l l d i s r u p t i o n , the b a c t e r i a l suspension i s placed in a s t e e l cylinder f i t t e d with a piston and a small nylon r e l i e f valve which i s connected to an outlet tube. The assembly i s put i n a hydraulic press and a pressure of ~1200 l b / i n 2 applied. The r e l i e f valve i s adjusted to give a slow steady flow. Breakage i s effected by the shear forces generated as the c e l l s flow out of the small o r i f i c e and by explosive decompression as the c e l l s experience a rapid, 1200 l b / i n 2 pressure drop. The other half of the c e l l p e l l e t prepared above ( i n 1) was resuspended i n 3 cm3 of pH 6.8 buffer to give a t o t a l volume of 5.2 cm3 and 0.1 mg DNAse added. The mixture was passed through a precooled (0°C) 58 French pressure c e l l at 1260 l b / i n 2 and c o l l e c t e d i n a cooled (0°C) container. The process was repeated three times. After c e n t r i f u g a t i o n for 15 minutes at 15000 rpm i n a SS-34 rotor the supernatant and p e l l e t (resuspended i n 10 cm3 pH 6.8 buffer) were both assayed. The t o t a l a c t i v i t y recovered (supernatant and p e l l e t ) was 91% of the o r i g i n a l a c t i v i t y . The a c t i v i t y associated with the supernatant was 70% of the recovered a c t i v i t y . C e l l breakage was therefore good. 3) Sonication 8 1* In this method of c e l l d isruption the rapid v i b r a t i o n of an u l t r a -sonic probe induces the formation of gas bubbles moving at high v e l o c i t y i n the v i c i n i t y of the t i p ('cavitation'). The high shear forces generated by these r a p i d l y moving bubbles disrupts the c e l l w a l l . The c e l l p e l l e t (about 160 g wet weight) from a 10 l i t r e l a t e log phase culture grown on L u r i a broth was thawed at 30°C and resuspended to a t o t a l volume of 245 cm3 i n pH 6.8 buffer to which 0.5 mg DNAse had been added. The mixture was s t i r r e d i n an ice bath and sonicated with the aid of a Bronwill Biosonik IV sonicator equipped with a 3/4" probe and set at high power. Sonication was c a r r i e d out in 6 one minute bursts with one minute of cooling between each burst. The mixture was then assayed and the t o t a l a c t i v i t y recovered was 98% of the o r i g i n a l . The mixture was centrifuged for 20 minutes at 9000 rpm i n a GS-3 rotor. The supernatant was assayed and found to contain 10% of the recovered a c t i v i t y . C e l l breakage was therefore poor. 59 4) B a l l i s t i c D i s i n t e g r a t i o n with Glass Beads 8 4 This method of c e l l d isruption r e l i e s on the v i o l e n t a g i t a t i o n of a c e l l suspension with small glass beads. The shear forces generated by the r a p i d l y moving beads disrupt the c e l l walls. A c e l l p e l l e t (prepared as for 3, above) was thawed at 30°C and resuspended to a t o t a l volume of 400 cm3 in pH 6.8 buffer to which 0.5 mg DNAse had been added. 100 g of 0.45-0.50 mm glass beads were added to the mixture. It was then blended in a precooled (0°C) three l i t r e Waring commercial blender at high speed i n two one minute bursts with one minute of cooling between the bursts. After centrifugation for 20 minutes at 9000 rpm i n a GS-3 rotor the supernatant was assayed and found to contain 6% of the t o t a l a c t i v i t y . It was not possible to resuspend the p e l l e t completely and thus ascertain the percentage recovery of a c t i v i t y . The c e l l breakage was therefore poor. These r e s u l t s are summarized below, Table IV. Table IV: Comparison of C e l l Breakage Techniques. Method Percentage A c t i v i t y Recovered Percentage C e l l Breakage (based on recovered a c t i v i t y ) Lysozyme and Freeze/Thaw 67% 23% French Pressure C e l l 91% 80% Sonication 98% 10% B a l l i s t i c D i sintegration - approx. 6% 60 N.B. a) Precooling was carried out i n the l a t t e r three techniques to minimise heating e f f e c t s associated with the shear forces generated. b) The DNA l i b e r a t e d from disrupted c e l l s caused the mixture to be both viscous and mucilaginous. It was d i f f i c u l t to handle and an attempt was made to pass the mixture through a 21 gauge needle i n order to break up the DNA by shear force. This was not p r a c t i c a l because of the volumes involved and DNAse had to be added i n order to hydrolyse the DNA polymer and thus reduce the v i s c o s i t y . The French pressure c e l l i s obviously the method of choice. For cultures from which 6-glucosidase i s o l a t i o n was attempted, the following methodology was followed. The c e l l p e l l e t was thawed at 30°C and resus-pended to a f i n a l volume of from 200-400 cm3 i n pH 6.8 buffer. 0.5 mg DNAse and 0.1 mg of the protease i n h i b i t o r 8 5 phenylmethylsulphonyl f l u o r i d e (PMSF) were added per 100 cm3 of c e l l suspension. The mixture was then sonicated twice, as described above, for 2.5 minutes. If the sonication step was omitted the nylon valve i n the pressure c e l l was found to be worn away aft e r processing only 30-50 cm3 of the c e l l suspension. The mixture was then twice passed through a 30 cm3, s e l f f i l l i n g , pre-cooled (0°C) French pressure c e l l at 1250 l b / i n 2 . This procedure and a l l subsequent procedures were c a r r i e d out at 0-4°C. The c e l l debris was removed by centrifugation at 9000 rpm for 40 minutes on a GS-3 r o t o r . The supernatant was assayed, the p e l l e t resuspended i n buffer and again centrifuged down. This 'washing' of the p e l l e t was repeated u n t i l the 61 amount of B-glucosidase act ivi ty liberated into the supernatant was less than 2% of that obtained from the f i r s t extraction. The supernatants were combined and this c e l l free extract was used immediately for subsequent isolation of 8-glucosidase. 62 II.3 P u r i f i c a t i o n and I s o l a t i o n of 8-Glucosidase from A. f a e c a l i s  and Related Details II.3.1 General Methods Ammonium sulphate used was Canadian S c i e n t i f i c Products u l t r a pure spe c i a l enzyme grade. 3-(N-Morpholino)-propanesulphonic acid (MOPS), triethanolamine, myo-inositol, streptomycin sulphate and c e l l o b i o s e were obtained from the Sigma Chemical company. Electrophoresis reagents were obtained from BioRad with the exception of sodium dodecyl sulphate which was obtained from the Sigma Chemical Company. A l l other chemicals not previously described were general purpose reagent grade. Buffers refer to 100 mM sodium phosphate buffers prepared as described (II.2.1) unless otherwise s p e c i f i e d . Buffers other than sodium phosphate buffers were prepared by back t i t r a t i o n of the buffering agent with either sodium hydroxide or hydrochloric acid as appropriate. A l l water used was double deionised (except FPLC grade water). U l t r a c e n t r i f u g a t i o n was c a r r i e d out on a Beckman L-350 ult r a c e n t r i f u g e equipped with a 45-TI rotor and ce n t r i f u g a t i o n was c a r r i e d out as previously described (II.2.1). DE-52 ion exchange r e s i n and Sephacryl S-200 gel chromatography r e s i n were packed and prepared, according to the manufacturers i n s t r u c t i o n s , i n LKB 2137 chromatography columns. Column dimensions are described i n the main body of the text. A l l columns (excluding FPLC columns) were run i n the cold room at 4°C. A constant flow rate was 63 maintained with a LKB 2132 Microperpex p e r i s t a l t i c pump. Eluent was monitored at 280 nm with a LKB 2138 Uvicord-S single beam UV monitor linked to a LKB 2210 single channel f l a t bed recorder. Fractions were co l l e c t e d with the aid of a LKB 2112 f r a c t i o n c o l l e c t o r . DE-52 i s a strong anion exchanger and the species immobilised i s diethylaminoethyl. The DE-52 column was cleaned after each use by flushing with two bed volumes of the appropriate buffer containing 10% sodium chloride followed by at least three bed volumes of buffer containing no sodium c h l o r i d e . After two or three runs a black band b u i l t up at the top of the column (p o s s i b l y contaminant from the water p u r i f i c a t i o n treatment) and r e s o l u t i o n decreased. When this happened the black portion of the packing material was discarded and the column repacked. Both the DE-52 and S-200 columns were stored i n s o l u t i o n containing 0.02% sodium azide (to prevent microbial growth) when not i n use for periods longer than one week. The fast protein l i q u i d chromatography (FPLC) system i s supplied by Pharmacia Fine Chemicals as a complete medium pressure chromatography unit optimized for use with MonoBead high r e s o l u t i o n packing materials. The columns are supplied prepacked and include a cation exchange column, Mono S, any anion exchange column, Mono Q, a chromatofocussing column, Mono P and a gel chromatography column, Superose 12. Because of the short run times involved i n the use of t h i s system (of the order of 30-180 minutes) runs were ca r r i e d out at room temperature. Water used was 64 d i s t i l l e d , double deionised, r e d i s t i l l e d and f i l t e r e d through M i l l i p o r e 0.22 um Fluoropore f i l t e r s . If simply double deionised and f i l t e r e d water was used an i n t r a c t a b l e brown band b u i l t up at the top of the column. A l l samples were f i l t e r e d through 0.22 |jm f i l t e r s before a p p l i c a t i o n to the column. Column regeneration and run procedures used were as recommended by the manufacturer. Fractions c o l l e c t e d were assayed for 8-glucosidase a c t i v i t y as follows. 10ul aliquots of 5mM PNPG i n 100 mM pH 6.8 sodium phosphate buffer were spotted onto white waxed paper (Benchcote) and 10 uL of each f r a c t i o n added. If p-glucosidase a c t i v i t y was present a yellow colour developed a f t e r between 30 seconds and 20 minutes. This assay was found to be s e n s i t i v e down to a least 0.025 units/cm 3. Following t h i s the enzyme a c t i v i t y i n those f r a c t i o n s containing p-glucosidase was quantified as described (II.2.1) with 5-10 uL samples. Units of a c t i v i t y expressed on chromatograms are increase i n absorbance measured at 400 nm i n 50 seconds i n a standard assay cuvette (II.2.1). The units of s p e c i f i c a c t i v i t y are units of p-glucosidase a c t i v i t y (previously defined II.2.1) per mg of protein. Protein was assayed with the aid of BioRad protein assay k i t (based on measured absorbance upon binding of the dye Coomasie blue) following the manufacturers i n s t r u c t i o n s , using bovine serum albumin (BSA) to construct a c a l i b r a t i o n curve. A l l assays were carried out i n duplicate with appropriate d i l u t i o n s of the sample being assayed. D i a l y s i s was c a r r i e d out i n the cold room at 4°C i n either 10 or 65 25 mm Spectrapor d i a l y s i s tubing with a molecular weight cut off of 12,000-14,000 Daltons. Prior to use the tubing was twice boiled i n double deionised water. Sodium dodecyl sulphate denaturing polyacrylamide gel e l e c t r o -phoresis (SDS-PAGE) was c a r r i e d out in a discontinuous Laemmli system as described by Hames 8 6 using a BioRad Protean dual v e r t i c a l slab gel electrophoresis c e l l and a BioRad 500/200 power supply. The slab gels prepared were 15% acrylamide, 1.5 mm thick and prepared according to Hames 8 6. The samples were d i l u t e d to allow loading of between 5 and 50 ug of protein and pre-treated as d e s c r i b e d 8 6 . The gels were developed through the stacking gel at 25 mA constant current and through the resolving gel at 50 mA constant current. In order to determine which band corresponded to 8-glucosidase some samples were not boiled during pre-treatment i n order to avoid complete denaturation and the following procedure applied. After the gel was run, and p r i o r to Coomasie blue s t a i n i n g , a piece of Whatman No.l f i l t e r paper which had been soaked i n 5 mM PNPG ( i n 100 mM pH 6.8 sodium phosphate buffer) was placed against the gel and incubated at 37°C. After 30 minutes yellow bands were v i s i b l e which corresponded to protein bands on the subsequently stained g e l . There was no difference i n band pattern between otherwise i d e n t i c a l boiled and unboiled samples. The gels were stained for protein with Coomasie blue dye as d e s c r i b e d 8 6 . Other materials and methods have been described previously. 66 II.3.2 P u r i f i c a t i o n and I s o l a t i o n of B-Glucosidase from A. f a e c a l i s The method used for the p u r i f i c a t i o n and i s o l a t i o n of 8-glucosidase from A. f a e c a l i s i s based on that of Han and S r i n i v a s a n 5 7 . The method was modified over eight attempts, two of which were successful. The successful procedure i s summarised below, F i g . 20. A discussion of unsuccessful techniques t r i e d , the r a t i o n a l e behind the modifications and the s l i g h t differences between the two successful procedures follows a detailed d e s c r i p t i o n of the method used for the p u r i f i c a t i o n of the 8-glucosidase on which most of the characterisation was c a r r i e d out. The c e l l p e l l e t from a l a t e log phase culture grown i n Luria broth (II.2.2) was thawed at 30°C and resuspended to a t o t a l volume of 330 cm3 i n pH 6.8 buffer and found to contain 1250 units of B-glucosidase a c t i v i t y . C e l l free extract was prepared as described (II.2.4) and a f t e r washing the p e l l e t twice 950 units were found to be associated with the combined supernatants and 28 units with the c e l l p e l l e t . The c e l l free extract (450 cm 3) was cooled to 0°C and a l l subsequent operations were carried out at 0-4°C with the exception of the FPLC steps (room tempera-ture) . To the s t i r r e d cooled solution streptomycin sulphate (6.75 g, 1.5% w/v) was slowly added and the s t i r r i n g continued for 15 minutes af t e r 67 f c e l l debris (discard) C e l l p e l l e t preparation of c e l l free extract p r e c i p i t a t e (discard) c e l l free extract 1.5% streptomycin sulphate supernatant 35-55% (NH 1 +) 2S0 1 + cut p r e c i p i t a t e - redissolve - dialyse - DE-52 column - S-200 column 'Mono-Q' column: -1) phosphate -2) triethanolamine - S-200 column pure 6-glucosidase Figure 20: Flow Chart for the I s o l a t i o n and P u r i f i c a t i o n of B-Glucosidase from A. F a e c a l i s . addition was complete. The mixture was centrifuged for 60 minutes at 35,000 rpm i n a TI-45 rotor and the pr e c i p i t a t e d DNA and RNA discarded. The supernatant, containing 903 units of a c t i v i t y , was di l u t e d to 610 cm3 with double deionized water. 68 Ammonium sulphate was ground to a fine powder with a pestle and mortar and 127.5 g slowly added to the s t i r r e d cooled solution to give a 35% saturated s o l u t i o n 4 4 . S t i r r i n g was continued for 30 minutes a f t e r addition was complete and the mixture then centrifuged for 25 minutes at 9000 rpm i n a GS-3 r o t o r . The p r e c i p i t a t e d protein p e l l e t was redissolved i n 100 cm pH 6.8 buffer and found to contain 54 units of a c t i v i t y . It was discarded. To the cooled s t i r r e d supernatant was slowly added a further 82 g ammonium sulphate to give a 55% saturated s o l u t i o n 4 4 . S t i r r i n g was continued for 30 minutes a f t e r addition was complete and the mixture again centrifuged for 25 minutes at 9000 rpm i n a GS-3 r o t o r . The supernatant, containing 13 units i n 610 cm3, was discarded. The p r e c i p i t a t e d protein p e l l e t was redissolved i n a minimum volume of 122 cm3 of 20 mM pH 7.4 buffer containing 10 ug/cm3 PMSF and found to contain 670 units of a c t i v i t y . This s o l u t i o n was dialysed against 8 l i t r e s of the same buffer for 13 hours at which time 683 units were found i n the 160 cm3 s o l u t i o n . The solution was centrifuged for 20 minutes at 9000 rpm i n a GS-3 rotor to remove r e s i d u a l undissolved contaminants and loaded onto a DE-52 ion-exchange column ( 55 cm x 2 cm2, pre-equilibrated with 20 mM pH 7.4 buffer) at 55 cm 3/hour. The sample was washed i n (at 55 cm 3/hour) successively, with 100 cm3 20 mM pH 7.4 buffer and 400 cm3 of the same buffer containing 1.0% sodium c h l o r i d e . The combined eluent from these washings contained 78 units of a c t i v i t y and was discarded. The column was washed with a further 500 cm3 of 20 mM pH 7.4 buffer containing 1.0% 69 sodium c h l o r i d e . The washings contained less than 1 unit of 8-glucosidase a c t i v i t y and most of the unbound and p a r t i a l l y bound protein appeared to have been washed off the column (as monitored by absorbance at 280 nm)• The column was developed with 20 mM pH 7.4 buffer containing 2.0% sodium chloride at a flow rate of 10 cm 3/hour. 3.3 cm3 f r a c t i o n s were co l l e c t e d and the eluent was monitored at 280 nm at 2 0D f u l l scale with a 3 mm pathlength flow through c e l l . The f r a c t i o n s containing most of the enzyme a c t i v i t y were combined ( f r a c t i o n s 33-40 i n c l u s i v e , F i g . 21), the tubes being rinsed with 20 mM pH 7.4 buffer. The f r a c t i o n s combined, containing 485 units of a c t i v i t y i n 23.7 cm3 were loaded (at 25 cm 3/hour) onto a Sephacryl S-200 gel chromatography column (57 cm x 5.3 cm2, pre-equilibrated with 20 mM pH 7.4 b u f f e r ) . The column was developed with 20 mM pH 7.4 buffer at 12 cm 3/hour. 4.0 cm3 f r a c t i o n s were c o l l e c t e d and the e f f l u e n t was monitored at 280 nm at 0.5 OD f u l l scale with a 3 mm pathlength flow through c e l l . The f r a c t i o n s containing most of the enzyme a c t i v i t y were combined ( f r a c t i o n s 33-43 i n c l u s i v e , F i g . 22), the tubes being rinsed with 20 mM pH 7.4 buffer. The f r a c t i o n s combined, containing 467 units of a c t i v i t y i n 35 cm3 were f i l t e r e d through a M i l l i p o r e Milex-GS 0.22 pm single use f i l t e r . The s o l u t i o n was s p l i t into four equal portions and each was run separately 70 F r a c t i o n § Figure 21: P r o t e i n / A c t i v i t y P r o f i l e for DE-52 Chromatography Step. 1 .5-1 1 ' ' 1 (-100 F r a c t i o n § Figure 22: P r o t e i n / A c t i v i t y P r o f i l e for F i r s t S-200 Chromatography Step. 71 Figure 23: P r o t e i n / A c t i v i t y P r o f i l e for Mono Q (Phosphate) Chromatography Step. J 1 i • / / / / tea gradient—/ I OD280 / I / A / Q—«ctivrty j L X • f i Y k . -. r-" ) / / ' \ 4 l'o 2 V, 1.1 4'° Figure 24: P r o t e i n / A c t i v i t y P r o f i l e for Mono Q (Triethanolamine) Chromatography Step. .0 i—••—i 1 1 1 1 1 1 r 24 26 28 30 32 34 36 38 40 F r a c t i o n # Figure 25: P r o t e i n / A c t i v i t y P r o f i l e for Second S-200 Chromatography Step. on the FPLC Mono Q anion exchange column (to avoid overloading the column). The samples were loaded onto the column (5.0 cm x 0.36 cm 2, pre-equilibrated with 20 mM pH 7.4 buffer) from a 10 cm3 Superloop at 2 cm 3/minute. The column was developed at 2 cm3/minute with a 0-5% sodium chloride gradient i n 20 mM pH 7.4 buffer and the eluent monitored at 280 nm at 1.0 0D f u l l scale with a 10 mm pathlength flowthrough c e l l . Protein p r o f i l e ( i e . absorbance at 280 nm), a c t i v i t y p r o f i l e and the fr a c t i o n s retained are shown below ( F i g . 23) for one run. The fr a c t i o n s containing most of the B-glucosidase were combined and the tubes rinsed with 20 mM pH 7.4 buffer. 73 The f r a c t i o n s combined, containing 249 units of a c t i v i t y i n 29 cm3 were loaded back onto the Mono Q column from a 50 cm3 Superloop at 2 cm3/minute and washed i n with 30 cm3 20 mM pH 7.4 triethanolamine buffer to r e e q u i l i b r a t e the column. The column was developed with a 0-5% sodium chloride gradient i n the triethanolamine buffer ( F i g . 24). The f r a c t i o n s containing most of the 8-glucosidase were combined and the tubes rinsed with 20 mM pH 7.4 buffer (sodium phosphate). The f r a c t i o n s combined, containing 177 units of a c t i v i t y i n 7.9 cm3 were loaded and run on a Sephacryl S-200 gel chromatography column as described above. The protein was eluted as a single symmetrical 'peak'. The s p e c i f i c a c t i v i t y p r o f i l e (enzyme activity/absorbance at 280 nm) together with the a c t i v i t y and protein ( i e . absorbance at 280 nm) p r o f i l e s are shown, F i g . 25. The f r a c t i o n s of highest s p e c i f i c a c t i v i t y were combined and t h i s material, containing 98 units of a c t v i t y i n 15.4 cm3 was used for most of the c h a r a c t e r i s a t i o n c a r r i e d out on B-glucosidase from A. f a e c a l i s . A p u r i f i c a t i o n table for the whole procedure i s shown below, Table V. 74 Table V: P u r i f i c a t i o n of B-Glucosidase from A. fa e c a l i s . P u r i f i c a t i o n Protein Volume Total Total S p e c i f i c P u r i f i c a t i o n Y i e l d step (mg/cm3) (cm 3) protein (mg) a c t i v i t y (units) a c t i v i t y (units/mg) (%) 1. Streptomycin sulphate ppt 409 610 25000 903 0.036 — (100) 2. 35-55% (NH^)2S0lt 109 122 13300 670 0.050 1.4 74 3. DE 52 7 23.7 166 485 2.92 58 54 4. S-200 1.6 35 56 467 8.34 2.9 52 5. Mono-0 Phosphate 0.42 29 12.1 249 20.6 2.5 28 6. Mono Q Triethanolamine 7.9 — 177 — — 20 7. S-200 0.20 15.4 3.1 98 31.6 1.5 11 N.B. a) Protein was assayed using a BioRad protein assay k i t with bovine serum albumin as standard. b) The high loss of a c t i v i t y at the Mono Q (phosphate) step was probably due to the large surface area of glass to which the enzyme was exposed. It i s not unusual for proteins to be denatured by exposure to glass surfaces. 8 8 c) The apparent high loss of a c t i v i t y at the f i n a l S-200 step was because only fract i o n s having the highest s p e c i f i c a c t i v i t y were taken. 75 AFTER STEP 1 2 3 4 r POSSIBLE HIGH KIL. WT. IMPURITY WL. WT. >ARKERS PHOSPHORYLASE b 7 7 Figure 26: SDS-PAGE Gel of Pure and P a r t i a l l y Pure 8-Glucosidase. E glucosidase 15 Volume (mis) artifact 18 A n a l y t i c a l Gel Chromatography of Pure 8-Glucosidase on Superose 12 Column. 76 The protein was shown to be e s s e n t i a l l y homogeneous by SDS-polyacrylamide gel electrophoresis, F i g . 26, and by a n a l y t i c a l gel chromatography on a MonoBead Superose 12 column, F i g . 27. The f i n a l preparation (6 and 9 ug protein loading) ran as one major band on a 15% acrylamide SDS gel with a small high molecular weight impurity band (marked on F i g . 26). The major protein band was shown to have a high B-glucosidase a c t i v i t y as described (II.3.1). Some of the cruder preparations were also run, F i g . 26. The conditions under which the gel was run are described elsewhere (II.3.1) as i s the determination of sub-unit molecular weight on the same gel (I I . 4 ) . The f i n a l preparation ran as a single symmetrical peak on the Superose 12 column with two a r t i f a c t s of low apparent molecular weight, F i g . 27. The B-glucosidase also had an apparent molecular weight lower than expected and th i s i s discussed elsewhere (II.4.2). The conditions are described i n II.4.2. Some other points that should be noted about t h i s procedure are as follows. 1: The a c t i v i t y 'shoulder' observed on both of the Mono Q FPLC chromatograms may be due to: a) unrelated contaminating protein with B-glucosidic a c t i v i t y , b) sub-units of the (possibly) oligomeric B-glucosidase, c) p a r t i a l l y hydrolysed B-glucosidase s t i l l r e t a i n i n g some a c t i v i t y or d) an a r t i f a c t of the column, a) i s u n l i k e l y as one of the other steps would most probably have resolved d i f f e r e n t proteins. It i s not easy to d i s t i n g u i s h between the l a t t e r three 77 p o s s i b i l i t i e s . 2: The 'breakthrough' of 78 units on loading the sample during the DE-52 step i s due to the i n t r i n s i c high Ionic strength of the protein sample and could have been reduced by further d i l u t i o n prior to this step. 3: A previous p u r i f i c a t i o n , i d e n t i c a l except for the reversal of the two Mono Q steps and the omission of the f i n a l S-200 step, yielded B-glucosidase which ran as a single band on SDS-PAGE and had a s p e c i f i c a c t i v i t y of 45.7 units/mg. ( c f . 31.6 units/mg). The reason for the higher s p e c i f i c a c t i v i t y and easier p u r i f i c a t i o n i s not obvious (see I I I ) . II.3.3 Method Development The ammonium sulphate 'cut' found to give the maximum a c t i v i t y was 35-55% and not 40-60% as r e p o r t e d 5 7 . This may be due to the use of d i f f e r e n t tables of ammonium sulphate saturation than used i n the reported p r e p a r a t i o n 5 7 . Published tables of ammonium sulphate saturation often give d i f f e r e n t values (dependent on the conditions under which they were measured). D i a l y s i s followed by ion exchange chromatography was chosen as the f i r s t step after ammonium sulphate p r e c i p i t a t i o n , rather than gel f i l t r a -t i o n , because although the l a t t e r technique lowers the io n i c strength concomitantly with e f f e c t i n g a p u r i f i c a t i o n , the resolution i s dependent on small sample volume. The reso l u t i o n i n ion exchange chromatography i s 78 e s s e n t i a l l y i n dependent of sample volume and w i l l , i n a d d i t i o n t o p u r i f i c a t i o n , e f f e c t a c o n c e n t r a t i o n of d i l u t e samples. The i o n exchange column was d e v e l o p e d i s o c r a t i c a l l y a f t e r i t was f o r t u i t o u s l y o b s e r v e d d u r i n g an e a r l y i s o l a t i o n a t t e m p t , when washing a sample i n a t 2% sodium c h l o r i d e (as d e s c r i b e d 5 7 ) , t h a t the 8 - g l u c o s i d a s e a c t i v i t y was e l u t e d w i t h r e s o l u t i o n of the p r o t e i n i n t o 'peaks' (as m o n i t o r e d a t 280 nm). T h i s s u g g e s t e d t h a t the B - g l u c o s i d a s e was p a r t i a l l y bound t o the column a t t h i s i o n i c s t r e n g t h and ' t r u e chromatography' ( p a r t i t i o n i n g between a m o b i l e and s t a t i o n a r y phase) was t a k i n g p l a c e . C o n d i t i o n s f o r i o n exchange chromatography on the FPLC Mono Q column were d e t e r m i n e d as f o l l o w s . The b u f f e r s shown were p r e p a r e d , T a b l e V I . W i t h the e x c e p t i o n o f 20 mM pH 7.4 sodium phosphate t h e s e a r e the b u f f e r s recommended by the m a n u f a c t u r e r f o r use w i t h the Mono Q column. A p a r t i a l l y pure B - g l u c o s i d a s e p r e p a r a t i o n ( s p e c i f i c a c t i v i t y = 10.1 u n i t s / m g ) c o n t a i n i n g 2.3 u n i t s / c m 3 i n 20 mM pH 7.4 sodium phosphate was d i v i d e d i n t o 0.5 cm 3 a l i q u o t s w h i c h were d i a l y s e d a g a i n s t 1 l i t r e o f each of t h e b u f f e r s ( e x c e p t pH 7.4 phosphate b u f f e r ) o v e r n i g h t . These a l i q u o t s were t h e n r u n w i t h a 0-5% l i n e a r sodium c h l o r i d e g r a d i e n t on t h e Mono Q column p r e - e q u i l i b r a t e d w i t h b u f f e r s o f the a p p r o p r i a t e pH. 79 Table VI; Buffers Used i n Determination of Conditions for Mono Q Chromatography. Buffering agent pH Ionic strength (mM) 3-(N-Morpholino)-propanesulphonic acid 6.60 50 •• 6.90 i* II 7.20 it Triethanolamine 7.40 ii •• 7.60 •• Sodium phosphate 7.40 20 •• 6.90 •• Run conditions were as recommended by the manufacturer. The samples run at pH 6.6 and 7.6 were completely inactivated i n d i c a t i n g that the enzyme is denatured at these pH's. The samples run at intermediate pH'S had e s s e n t i a l l y i d e n t i c a l chromatographic p r o f i l e s , the greatest difference being between the samples run at pH 7.4 i n triethanolamine and sodium phosphate b u f f e r s . I t was therefore decided to carry out the separation in the l a t t e r two buffers. II.3.4 Enzyme Storage A pure B-glucosidase sample with a s p e c i f i c a c t i v i t y of 45.7 units/mg containing 18.3 units/cm 3 i n pH 7.4 20 mM buffer was s p l i t into two equal portions. One portion was made up to 1% w/v with myo-inositol, 80 frozen i n l i q u i d nitrogen, l y o p h i l i s e d and stored i n the freezer at -20°C. 1% w/v myo-inositol was suggested to give complete protection of a c t i v i t y against l y o p h i l i s a t i o n 5 7 . The other portion was stored i n solution under toluene vapour at 4°C i n the r e f r i g e r a t o r . After two months the l y o p h i -l i s e d sample was made up to 1.0 cm3 i n 20 mM pH 7.4 buffer and assayed. It was found to contain 62% of the o r i g i n a l a c t i v i t y . The sample stored under toluene vapour was found to contain 83% of the o r i g i n a l a c t i v i t y . Myo-inositol showed no measurable i n h i b i t i o n of 8-glucosidase a c t i v i t y at 2% w/v. Storage under toluene vapour appears to be the best method of medium term storage. II.3.5 Unsuccessful P u r i f i c a t i o n Techniques Attempted 1) Mono S Cation Exchange Chromatography A p a r t i a l l y pure 8-glucosidase preparation (2.0 cm3) was dialysed against 1 l i t r e of 10 mM pH 6.7 (the lowest pH at which the enzyme i s stable) buffer and applied to a Mono S cation exchange column (5.0 cm x 0.36 cm2, pre-equilibrated with 20 mM pH 6.7 b u f f e r ) . The enzyme was eluted i n one bed volume of start b u f f e r . This method was therefore of no u t i l i t y i n the p u r i f i c a t i o n of 8-glucosidase. 81 2) Chromatic-focussing A pure 6-glucosidase sample (as described above, II.3.2) was d i a -lysed against the start buffer recommended by the manufacturer for chroma-tofocussing on a FPLC Mono P column from pH 7-4. The column was developed according to the manufacturers Instructions. A single symmetrical protein peak (monitored at 280 nm) was eluted at pH ~ 5 No 8-glucosidase a c t i v i t y was eluted at any pH (assays carried out at pH 7). 3) A f f i n i t y Adsorption Chromatography Approximately 1 cm3 of Pierce Se l e c t i n 13 a f f i n i t y support was packed i n a Pasteur pipette plugged with glass wool. This material con-s i s t s of ce l l o b i o s e immobilised, with no spacer arm, onto a s o l i d support matrix of agarose at a concentration of 35-40 pmoles/cm3 g e l . This column was equil i b r a t e d with 20 mM pH 7.4 buffer and a pure 6-glucosidase sample (as described above, II.3.2) containing 9 units of a c t i v i t y was applied. A l l of the a c t i v i t y was eluted i n one bed volume of start b u f f e r . This method was therefore of no u t i l i t y i n the p u r i f i c a t i o n of B-glucosidase. 4) A f f i n i t y E l u t i o n Chromatography It was hoped that addition of 8-glucosidase substrates to the developing buffer of an ion exchange column would s p e c i f i c a l l y elute the 82 B-glucosidase by deformation and/or n e u t r a l i s a t i o n of charge at the active s i t e . A p a r t i a l l y pure 8-glucosidase sample containing 186 units i n 75 cm3 of 20 mM pH 7.4 buffer and with a s p e c i f i c a c t i v i t y of 16 units/mg was loaded onto a DE-52 column (55 cm x 2 cm2, pre-equilibrated with 20 mM pH 7.4 buffer) as described (II.3.2). The sample was washed in with 1% sodium chloride in star t b u f f e r . After a l l unbound protein had been eluted the column was developed with wash buffer which had been made up to 20 mM with c e l l o b i o s e . No protein (monitored at 280 nm) or B-glucosidase a c t i v i t y was eluted i n 500 cm 3. The column was then developed with 20 mM pH 7.4 buffer containing 0.68% sodium chloride and 0.32% 8-D-gluco-pyranosyl pyridinium bromide ( t o t a l s a l t concentration = 1%). No protein or B-glucosidase a c t i v i t y was eluted i n 500 cm 3. Comparison of the UV spectra of the buffer before and a f t e r passage through the column, F i g . 28, indicated that no appreciable hydrolysis of B-D-glucopyranosyl pyridinium bromide had taken place. Development of the column with buffer containing 0.32% 8-D-glucopyranosyl pyridinium bromide and 1.18% sodium chloride eluted a small amount of B-glucosidase a c t i v i t y i n a large volume (~ 200 cm 3). This had previously been observed during development with buffer containing 1.5% sodium c h l o r i d e . The 8-glucosidase was f i n a l l y eluted with buffer containing 1.68% sodium chloride and 0.32% B-D-glucopyranosyl pyridinium bromide. Therefore addition of 8-glucosidase substrates to the developing buffer was no improvement on the use of buffers containing sodium chloride alone. 83 Figure 28: UV Spectra of A f f i n i t y E l u t i o n Buffer Before and A f t e r Passage Through A DE-52 Column Containing 8-Glucosidase. 84 II.4 Characterisation of p-Glucosidase from A. Faecalis II.4.1 General Methods A l l k i n e t i c measurements were made i n 100 mM pH 6.8 sodium phosphate buffer at 37°C with the exception of glucosyl f l u o r i d e which was made i n triethanolamine buffer. The B-glucosidase used was that prepared as described i n d e t a i l (II.3.2). Water used was double deionised. A l l substrates and i n h i b i t o r s not synthesised or obtained as already described were obtained from the Sigma Chemical Company with the following exceptions. 2,4-Dinitrophenyl-p-D-glucopyranoside was prepared by Dr. Paul Bird (UBC). C e l l o t r i o s e , c e l l o t e t r a o s e and cellopentaose were prepared by Ne i l Gilkes (UBC). A l l volumes were measured with grade A a n a l y t i c a l volumetric glassware and/or Hamilton M i c r o l i t r e syringes and weights were measured on micro or semi-micro balances to at least four decimal places. A l l samples were pre-equilibrated to 37°C ± 0.1°C for at least 10 minutes i n quartz or glass cuvettes i n a Pye-Unicam PU-8800 u l t r a - v i o l e t / v i s i b l e recording spectrophotometer attached to a Julabo VI c i r c u l a t i n g thermostat bath unless otherwise stated. Reaction was i n i t i a t e d by addition of between 20 and 50 uL of an appropriate d i l u t i o n of 8-glucosidase stock so l u t i o n unless otherwise stated. 85 I n i t i a l reaction rates were, with the exception of the f l u o r i d e , determined by measuring the rate of change of absorbance of the reaction mixture at an appropriate wavelength In the PU-8800 spectrophotometer. E x t i n c t i o n c o e f f i c i e n t s were determined at the appropriate wavelengths, i n duplicate at 37°C i n 100 mM pH 6.8 sodium phosphate buffer for p-D-glucopyranosyl pyridinium bromide/pyridine ( d i s t i l l e d from potassium hydroxide under nitrogen) (Ae = -2925), p-nitrophenol ( r e c r y s t a l l i s e d from 0.01 M hydrochloric acid) (Ae = 7280) and 2,4-dinitrophenol ( r e c r y s t a l l i s e d from 0.01 M hydrochloric acid) (AE = 11,300). The e x t i n c t i o n c o e f f i c i e n t for 2,4-thiodinitrophenol was determined (at 400 nm) by allowing a reaction mixture containing 2.956 mM 2,4-dinitrothiophenyl - B-D-glucopyranoside to react for 24 hours i n the presence of p-glucosidase (Ae = 1698). The v a r i a t i o n of e x t i n c t i o n c o e f f i c i e n t with temperature (at 400 nm) for p-nitrophenol was also determined, F i g . 29. 40-- 3 0 -B 20-c k-l> D-E " 10-4500 5000 5500 6000 6500 7000 7500 Extinction coefficient Figure 29: V a r i a t i o n of PNP Ex t i n c t i o n C o e f f i c i e n t With Temperature at pH 6.8. 86 A l l other materials and methods not previously described are described i n the main body of the text. II.4.2 Molecular Weight Determination Sub-unit molecular weight was determined by running a Sigma MW-SDS-200 k i t on the SDS-PAGE gel previously described (II.3.1) i n a track adjacent to the 8-glucosidase. The proteins, sub-unit molecular weights and RF's i n the 15% sodium dodecyl sulphate gel are shown below, Table VII. Table VII; Molecular Weight Markers for SDS-PAGE. Protein Sub-unit Rf Molecular weight (Daltons) Phosphorylase b 97,000 0.16 Bovine serum albumin 67,000 0.27 Ovalbumin 43,000 0.45 Carbonic anhydrase 30,000 0.68 Trypsin i n h i b i t o r 20,100 0.86 a-Lactalbumin 14,400 0.99 B-Glucosidase from A. f a e c a l i s 51,500 (determined) 0.40 87 A plot of log (molecular weight) against RF yielded a straight l i n e from which the sub-unit molecular weight of the unknown protein could be determined, F i g . 30. An attempt was made to determine the oligomeric molecular weight by using a MonoBead Superose 12 gel chromatography column i n conjunction with the FPLC system. Samples (50 uL) were run at 0.25 cm3/minute i n 100 mM pH 6.8 sodium phosphate buffer. Blue dextran (a high molecular weight poly-saccharide) and acetone were run to determine the exclusion volume and void volume of the column r e s p e c t i v e l y . The following proteins from a Sigma non-denatured protein molecular weight marker k i t were also run, Table VIII. Table VIII; Molecular Weight Markers for Superose 12 Gel Chromatography. Protein Molecular weight Rf (Daltons) Bovine serum albumin (dimer) 132,000 1.37 Bovine serum albumin (monomer) 66,000 1.52 Phosphoglucomutase 61,600 1.61 Ovalbumin nhydrase 45,000 1.64 a-Lactalbumin 14,200 1.82 3-Glucosidase from A. f a e c a l i s 75,000 (determined) 1.50 Phosphorylase b 194,000 1.44 A logarithmic plot of molecular weight against RF yielded a straight l i n e from which the molecular weight of the unknown protein was determined, F i g . 31. The molecular weight determined for the 8-glucosidase was lower 88 Rf Figure 3 1 : Logarithmic Plot of Molecular Weight Against R f For Superose 1 2 Gel Chromatography. 89 than expected 5' at 75,000 Daltons. The apparent molecular weight of phosphorylase b was 100,000 Daltons and not the known dimeric weight of 194,000 Daltons. Both phosphorylase and 8-glucosidase bind o l i g o -saccharides and i t i s probable that both are retarded on the agarose based support of the Superose 12 column, leading to a r t i f i c i a l l y low apparent molecular weights. A l l runs showed two a r t i f a c t u a l low molecular weight peaks at the same Rf's. The reason for th i s i s not c l e a r . II.4.3 Ki n e t i c s 1:Substrates To measure the i n i t i a l rate of reaction i t i s necessary to measure the rate of production of one or more i n i t i a l products. Three approaches to t h i s measurement were used i n t h i s study: 1) The rate of change of absorbance was monitored at an appropriate wave-length where the substrate and one of the i n i t i a l products had s u f f i c i e n t -l y d i f f e r e n t e x t i n c t i o n c o e f f i c i e n t s . 2) I f the ex t i n c t i o n c o e f f i c i e n t s were not s u f f i c i e n t l y d i f f e r e n t at an accessible wavelength a coupled assay was used. One of the i n i t i a l products (glucose) was a substrate for an enzyme catalysed reaction(s) which produced a measurable change i n absorbance. 3) The rate of production of the i n i t i a l product of the hydrolysis of 8-D-glucopyranosyl f l u o r i d e ( f l u o r i d e ion) was monitored by an i o n -s p e c i f i c electrode. A l l i n i t i a l rates were measured at less than 5% reaction ( l i n e a r 90 portion), with the exception of the fluoride which was measured as described in the text. Wavelengths or methods by which reactions were monitored are shown below, Table IX. Table IX: 8-Glucosidase Substrates. Substrate Cuvette type Wavelength Reference ( a l l 8-linked) and pathlength (mm) (nm) p-nitrophenyl-8-D-glucopyranoside 10 400 10 p-nitrophenyl-8-D-galactopyranoside 10 400 18 p-nitrophenyl-8-D-mannopyranoside 10(m) 400 18 8-D-glucopyranosyl azide 10(m) 245-K; 87 B-D-glucopyranosyl pyridinium bromide 2 265 17 2,4-dinitrophenyl-8-D-glucopyranoside 10 400 — 2,4-dinitrophenyl-8-D-l-thioglucopyranoside 10(m) 400 — 8-D-glucopyranosyl fluoride — electrode — cellobiose 10(sm) G — cellotriose 10(m) G — cellotetraose I0(m) G — cellopentaose 10 (m) G — lactose 10(sm) G — sucrose 10(sm) G — sa l i c in 10(sm) G — gentobiose 10(sm) G — 91 N.B. a) m indicates that micro cuvettes were used (absorbance could be measured on a volume as low as 0.22 cm3) and sm designates that semi-micro cuvettes were used (nominal 1.0 cm 3). A l l other cuvettes used held a nominal 3 cm 3. b) G indicates that a coupled glucose assay (described i n text) was used to measure the rate of release of glucose. c) Electrode Indicates that an ion s p e c i f i c electrode was used to measure the rate of hydrolysis of this substrate. d) 0.25-0.50% Bovine serum albumin (Sigma A 7906 or A 4378) was added to a l l reaction mixtures (except the f l u o r i d e , XVII). If bovine serum albumin was not added, a time dependent loss of enzyme a c t i v i t y was observed on d i l u t i o n into the cuvette ( p a r t i c u l a r l y when d i l u t e enzyme stock s o l u t i o n was used)• This loss of a c t i v i t y i s due to denaturation of enzyme on the glass surface and has been observed b e f o r e 8 8 with other 8-glucosidases at high d i l u t i o n . A comparison of k i n e t i c parameters i n the presence and absence of bovine serum albumin was made with PNPG and no difference was found. e) A l l 8-glucosidase d i l u t i o n s were made into 100 mM pH 6.8 sodium phosphate buffer containing 1% bovine serum albumin. f) Before measuring k i n e t i c parameters the p-glucosidase stock s o l u t i o n was standardised by measuring i t s a c t i v i t y against a reference of 1.34 mM PNPG ( i n duplicate) under the conditions used for determination of the k i n e t i c parameters of PNPG. The exceptions are PNPGal and PY-G which had the i r k i n e t i c parameters 92 determined using stock enzyme solutions that had been used for a f u l l determination of the k i n e t i c parameters of PNPG. The coupled glucose assay used was based on the following reaction: . » m r . hexokinase „ , „ . Glucose + ATP • G-6-P + ADP G-6-P + NADP G ~ 6 ~ P D > 6-PG + NADPH where: ATP i s adenosine triphosphate, G-6-P i s glucose-6-phosphate, NADP i s the cofactor B-nicotinamide adenine dinucleotide phosphate, G-6-PD i s glucose-6-phosphate dehydrogenase, 6-PG i s 6-phosphogluconic acid, and NADPH i s the reduced form of NADP. The e x t i n c t i o n c o e f f i c i e n t difference NADPH-NADP at 340 nm i s 6220 and the release of glucose can be quantified by the increase i n absorbance at this wavelength. The assay mixture used was: G-6-PD Sigma G-5760, 45 \iL containing 90 units of a c t i v i t y ; Hexokinase Sigma H-5875, 37 |iL containing 150 units of a c t v i t y ; 0.3 cm3 12 mM ATP Sigma A-2383, i n water; 0.3 cm3 7 mM NADP Sigma N-0505, i n water; 0.3 cm3 20 mM MgCl 2 (AR), i n water; and 0.6 cm 3 100 mM pH 6.8 sodium phosphate buffer, d i l u t e d no more than 4.3 times i n the reaction mix. These proportions are based on those found i n the Sigma 16-UV glucose assay. 93 In order for a coupled assay to e f f e c t i v e l y r e f l e c t the rate of glucose release the a u x i l i a r y enzymes (G-6-PD and hexokinase) must be present i n s u f f i c i e n t excess. To determine this excess the concentration of assay mix in a standard reaction mixture containing 1.34 mM ce l l o b i o s e was increased u n t i l further increases i n concentration did not increase the apparent i n i t i a l reaction rate; i . e . u n t i l the i n i t i a l rate observed was dependent on the rate of hydrolysis of cel l o b i o s e by 8-glucosidase and independent of the concentration of the a u x i l i a r y enzymes. On addition of 8-glucosidase to the reaction mixture there was an immediate l i n e a r increase i n absorbance and no 'lag phase' could be detected. To further confirm that the glucose assay was working as expected i t was used with a reaction mixture containing 0.137 mM PNPG. After measuring the r e l a t i v e contributions of NADPH and p-nitrophenol to absorbance at 340 and 400 nm the following i n i t i a l reaction rates were measured: based on glucose assay, 1.752 nmoles/minute based on p-nitrophenol release, 1.691 nmoles/minute. It should also be noted that pre-incubation of reaction mixtures containing the glucose assay removes any res i d u a l glucose present before hydrolysis i s i n i t i a t e d by addition of p-glucosidase. This assay has been developed independently 2 9. The k i n e t i c parameters of p-D-glucopyranosyl f l u o r i d e were determined as follows. An old Orion Ionalyzer f l u o r i d e ion s p e c i f i c electrode was regenerated 8 9. The electrode and an Orion single junction reference electrode were attached to a Radiometer PHM-82 d i g i t a l pH meter 94 set to measure m i l l i v o l t s . The pH meter was attached to a LKB 2210 sing l e channel flatbed recorder i n series with a variable m i l l i V o l t supply (to 'zero' the recorder). Solutions were preequilibrated to 37°C i n a polythene beaker i n a jacketted glass beaker attached to a Julabo VI c i r c u l a t i n g thermostat bath. It was confirmed that the B-glucosidase had the same a c t i v i t y (against PNPG) i n 100 mM pH 6.8 triethanolamine buffer as i n sodium phosphate buffer and a l l measurements were made i n triethanolamine buffer containing no BSA. Phosphate ions decrease the s e n s i t i v i t y of the electrode and i t was f e l t that high protein concentrations might also decrease i t s s e n s i t i v i t y . The system was cal i b r a t e d with 0-360 uM sodium f l u o r i d e at 37°C by standard additions, both before and after the k i n e t i c runs, eg. F i g . 32. The k i n e t i c parameters were determined by subtraction of the uncatalysed rate of 103-d 1 1 1 1 1 \r IU "I 1 1 1 1 1 h 180 200 220 240 260 280 300 - M i l l i v o l t s Figure 32: C a l i b r a t i o n Curve For Fluoride Electrode. 95 hydrolysis of B-D-glucopyranosyl f l u o r i d e from the 8-glucosidase catalysed rate for each sample. The substrate concentration at which the rate was measured was calculated by reference to the c a l i b r a t i o n curve. There was a s h i f t of ~18 mV between the c a l i b r a t i o n curves measured before and aft e r the k i n e t i c run. This corresponds to a doubling of concentration and the re s u l t s obtained may therefore be out by a factor of two. Attempts to determine the k i n e t i c parameters of p-nitrophenyl-8-D-c e l l o b i o s i d e as a substrate of B-glucosidase by monitoring the release of p-nitrophenol (as measured by increase i n absorbance at 400 nM) yielded unusual r e s u l t s . The i n i t i a l reaction rates were not l i n e a r but increased r a p i d l y as the reaction proceeded and the rates observed decreased as substrate concentration was increased. In an attempt to explain these observations the i n h i b i t i o n constant, K i , of th i s compound against PNPG as substrate was determined (see 'Inhibitors' below). In addition the products of the hydrolysis of p-nitrophenyl-B-D-c e l l o b i o s i d e by 8-glucosidase were analysed by paper chromatography as follows. It was confirmed that B-glucosidase was as active (against PNPG) in a v o l a t i l e 50 mM pH 6.8 triethylamine buffer as i n a 100 mM sodium phosphate buffer. Two 3 cm3 aliquots of 1.43 mM p-nitrophenyl-B-D-c e l l o b i o s i d e i n triethylamine buffer containing 0.3% BSA were hydrolysed (by B-glucosidase) to 10% and 20% completion respectively (measured by monitoring the release of p-nitrophenol as determined by absorbance at 400 nm). The reactions were stopped by heating at 100°C for 1 minute. A blank containing no B-glucosidase was also heated at 100°C for 1 minute 96 and no p-nitrophenol was released (as determined by absorbance at 400 nm)• The samples were repeatedly frozen ( i n a dry ice/acetone bath) and l y o p h i l i s e d to remove the v o l a t i l e buffer. The samples were then redissolved i n 40 uL water and 2.5 uL of each sample applied to an 1 m length of Whatman #1 f i l t e r paper together with 2.5 ug samples of glucose and c e l l o b i o s e i n order to carry out descending paper chromatography. The chromatogram was developed with ethyl acetate:acetic acid:formic acid:water 18:3:1:4 for 15 hours. The chromatogram was v i s u a l i s e d by dipping i t successively into solutions of 0.1% s i l v e r n i t r a t e i n acetone, 2% ethanolic potassium hydroxide and 5% aqueous sodium thiosulphate. This method of v i s u a l i s a t i o n i s s p e c i f i c for reducing sugars and did not v i s u a l i s e unreacted glycosides. Only glucose was found i n the samples (and no c e l l o b i o s e , RF r e l a t i v e to glucose = 0.43). Kin e t i c parameters for PNPG with a B-glucosidase preparation that had been stored under toluene vapour for three months were also determined and compared with the parameters determined using the fresh preparation. Km was i d e n t i c a l within experimental errors. With PNPG and PNPGal, substrate i n h i b i t i o n was observed and quantified at high substrate concentrations. Where substrate i n h i b i t i o n was observed with other substrates i t was not quant i f i e d . 2 i n h i b i t o r s Inhibitors of B-glucosidase were quantified against PNPG as sub-strate at f i v e substrate concentrations and four i n h i b i t o r concentrations (plus one determination with no i n h i b i t o r ) . K i n e tic parameters were 97 accurately determined for the following i n h i b i t o r s : B-D-glucosylamine, a-D-glucose, p-nitrophenyl-a-D-glucopyranoside, p-nitrophenyl-B-D-c e l l o b i o s i d e and D-glucono-S-lactone. The l a t t e r i n h i b i t o r was ra p i d l y hydrolysed to D-gluconic acid under the reaction conditions used. In order to minimise t h i s hydrolysis concentrated stock solutions were made up i n water immediately p r i o r to use and kept i n an ice bath and 20-40 pL were added to the reaction mixture immediately p r i o r to i n i t i a t i o n of hydrolysis by addition of B-glucosidase. A l l other i n h i b i t o r s were pre-equ i l i b r a t e d at 37°C i n the reaction mixture prior to addition of the B-glucosidase. The approximate i n h i b i t i o n constants of cel l o b i o s e and p-nitrophenyl-B-D-mannopyranoside (PNPMan) were determined at four i n h i b i t o r concentrations and a single substrate concentration i n order to demonstrate that hydrolysis of these compounds was taking place at the same s i t e or a s i t e i n t e r a c t i n g with that at which PNPG i s hydrolysed. II.4.4 Miscellaneous Characterisation l : E f f e c t of 8-Glucosidase Concentration The l i n e a r i t y of i n i t i a l reaction rate with B-glucosidase concen-t r a t i o n was checked. I n i t i a l reaction rate for a reaction mixture containing 1.34 mM PNPG run under standard conditions was plotted against 8-glucosidase concentration over the range of concentrations used. The re l a t i o n s h i p was l i n e a r . 2:Temperature Dependence The temperature dependence of the hydrolysis of PNPG was measured 98 between 5 and 45°C at f i v e substrate concentrations and the a c t i v a t i o n energy determined. 3:Metal Ion S e n s i t i v i t y The divalent metal ion dependency of the 8-glucosidase was investigated as follows. 1 cm3 of the stock 8-glucosidase so l u t i o n was dialysed against 1 l i t r e of 20 mM pH 7.4 sodium phosphate buffer containing 10 mM EDTA for 40 hours with two changes of buffer. K i n e t i c parameters were determined for PNPG at four substrate concentrations. One determination was ca r r i e d out i n the presence of 10 mM EDTA and the other i n the presence of 5 mM magnesium chloride; both determinations were ca r r i e d out i n the absence of bovine serum albumin. The k i n e t i c parameters were i d e n t i c a l within experimental erro r . The s e n s i t i v i t y of the B-glucosidase to various metal Ions was determined i n 50 mM pH 6.8 triethanolamine buffer i n order to avoid p r e c i p i t a t i o n of insoluble metal phosphates. The a c t i v i t y of the p-glucosidase was measured at 0.998 mM (~14 Km) and 0.0832 mM (~1 Km) PNPG at 1.6 mM metal ion concentration. The following a n a l y t i c a l grade metal s a l t s were used: ferrous ch l o r i d e , calcium chloride, manganese(II) ch l o r i d e , mercuric ch l o r i d e , cupric chloride and zinc sulphate. EDTA treated p-glucosidase was used. When magnesium ion was added to the p-glucosidase prior to the determination d i f f e r e n t r e s u l t s were obtained (see I I I ) . A l l determinations were carried out i n the absence of bovine serum albumin. 99 4:The Anomeric Configuration of the I n i t i a l Product The anomeric configuration of the i n i t i a l product of hydrolysis was determined according to the method of B a r n e t t 9 0 with 8-D-gluco-pyranosyl f l u o r i d e as substrate as follows. Solutions of a-D-glucose (91 mg/100 cm 3), 8-D-glucose (85mg/cm3) i n 5mM pH6.8 sodium phosphate buffer and 8-D-glucopyranosyl f l u o r i d e i n 20 mM pH 6.8 sodium maleate buffer were prepared. Immediately a f t e r preparation they were placed i n a polarimeter m i c r o c e l l (1 cm3 volume, 10 cm pathlength) and the o p t i c a l r o t a t i o n monitored (at the wavelength of the sodium D l i n e ) for 15-20 minutes i n a Perkin-Elmer 141 polarimeter. 40 p i °f 1M sodium carbonate was then added to the c e l l (and well mixed with the aid of a Pasteur pipette) to catalyse the mutarotation of any free sugar present, and the o p t i c a l r o t a t i o n again monitored. After confirming that 8-glucosidase retains a c t i v i t y i n sodium maleate buffer the process was repeated on a blank containing 0.2 cm3 of stock enzyme solu t i o n and 0.8 cm3 sodium maleate buffer. No change i n o p t i c a l r o t a t i o n was detected on addition of the sodium carbonate. The process was then repeated on a mixture consisting of 0.2 cm3 of stock enzyme solution and 0.8 cm3 of 14 mM 8-D-glucopyranosyl f l u o r i d e i n 20 mM pH 6.8 sodium maleate buffer. The anomeric configuration of the f i r s t product of hydrolysis was in f e r r e d from the d i r e c t i o n of the change i n o p t i c a l r o t a t i o n on addition of the sodium carbonate (see I I I ) . 100 II.5 Treatment of Data A l l values of Km and Vm, together with the errors associated with the scatter of the data, were calculated by f i t t i n g data to the normal, non-linear form of the Michaelis-Menten equation by the procedure of W i l k i n s o n 9 1 . This procedure was ca r r i e d out with the aid of a program written for an Apple II computer i n col l a b o r a t i o n with I. Street (UBC) and l i s t e d i n Appendix 2. The reported values of Vm are for a standard amount of B-glucosidase; that amount necessary to hydrolyse 10 nmoles/minute of PNPG at saturating PNPG concentration. Reference rates were measured at 1.34 mM PNPG. The actual rate of saturating PNPG hydrolysis (Vm) was calculated assuming Km (PNPG) = 0.083 and i s reported as 'enzyme concentration'. The data were plotted according to Lineweaver and Burke as shown i n Appendix 3. Data points showing substrate i n h i b i t i o n were not considered i n the determination of Km and Vm. Approximate values of i n h i b i t i o n constants (Ki) were determined by measuring i n i t i a l reaction rates at one substrate concentration (~ 1 mM PNPG) and 4 or 5 i n h i b i t o r concentrations and p l o t t i n g the r e s u l t s according to Dixon . For those i n h i b i t o r s which were not also substrates These methods and terms and/or th e i r derivation are explained i n Appendix 1. 101 (fo r which Km and Vm were determined) the i n h i b i t i o n constants (Ki) were accurately determined by measuring I n i t i a l reaction rates at f i v e substrate (PNPG) concentrations, i n the absence of i n h i b i t o r and at four i n h i b i t o r concentrations (giving 25 data points i n a l l ) . The data were plotted according to Lineweaver and Burke i n order to determine the * i n h i b i t i o n pattern (eg. competitive, non-competitive etc.) . The i n h i b i t i o n constant (Ki) for competitive i n h i b i t o r s was deter-mined by p l o t t i n g the apparent Km (determined by the procedure of Wilkinson against i n h i b i t o r concentration . The best s t r a i g h t l i n e was found by li n e a r regression analysis and the c o r r e l a t i o n c o e f f i c i e n t , R, i s reported. The i n h i b i t o r a-D-glucose showed p a r t i a l l y non-competitive k i n e t i c s ; replots of both slope and apparent Vm were hyperbolic. Secondary r e p l o t s 9 2 yielded values for the k i n e t i c constants. The best straight l i n e s for the secondary replots were found by li n e a r regression a n a l y s i s . The i n h i b i t o r p-nitrophenyl-a-D-glucopyranoside showed non-linear k i n e t i c s from which i t was not e a s i l y possible to determine k i n e t i c constants. Substrate i n h i b i t i o n by the substrates PNPG and PNPGal was quantified by p l o t t i n g r e c i p r o c a l i n i t i a l reaction rates against substrate * concentration . The values of Ki were determined by 'inspection' of the * These methods and terms and/or t h e i r derivation are explained i n Appendix 1. 102 obviously i n h i b i t e d data points by l i n e a r regression a n a l y s i s . Since non-weighted l i n e a r regression Is not v a l i d i n this case the analysis merely constitutes a convenient straight l i n e . A l l graphical plots related to i n h i b i t i o n (with the exception of the approximate Ki determinations) are shown in Appendix 3. Arrhenius plots were analysed by l i n e a r regression (neglecting the obviously non-linear point at the highest temperature) and the c o r r e l a t i o n c o e f f i c i e n t , R, i s reported. 103 CHAPTER III Results and Discussion 104 I I I . l P u r i f i c a t i o n (and Comparison with Previous Work) Three schemes for the p u r i f i c a t i o n of 8-glucosidase from A. f a e c a l i s have been previously p u b l i s h e d 5 7 • 5 8 > 6 0 , a l l by Srinivasan and co-workers. The scheme published with Bumm58 i s a s l i g h t modification of that published with Han 5 7. Although these schemes claim to have produced pure material, both the low s p e c i f i c a c t i v i t y 5 7 , and also the p r o t e i n / a c t i v i t y p r o f i l e of the f i n a l chromatographic s t e p 5 8 , suggest that this i s not so. E.O. Smith, i n a thesis submitted under the supervision of Srinivasan, p u r i f i e d the 8-glucosidase from A. f a e c a l i s to a s p e c i f i c a c t i v i t y f i f t y times that reported by Han and S r i n i v a s a n 5 7 , and stated that the material prepared p r e v i o u s l y 5 7 * 5 8 was impure. The pure material was prepared ( i n minute quantities) by E.O. Smith 6 0, u t i l i z i n g the unwieldy and inconvenient technique of d i s c - g e l electrophoresis. The s p e c i f i c a c t i v i t y of the material prepared during the course of t h i s work (31.6 or 45.7 units/mg of protein at pH 6.8 and 37°C) i s i n reasonable agreement with that r e p o r t e d 6 0 (55 units/mg of protein at pH 6.5 and 40°C) considering the differences i n pH, temperature and protein assay method used to determine s p e c i f i c a c t i v i t i e s . The difference i n s p e c i f i c a c t i v i t i e s between the material obtained from the two successful p u r i f i c a t i o n attempts (31.6 and 45.7 units/mg of protein) i s not e a s i l y explained. However, considering that both preparations ran as greater than 99% of a single band on SDS-PAGE i t 105 i s probable that the preparation of lower specific act iv i ty consisted mainly of one protein, a portion of which was denatured (inactive) while s t i l l retaining the physical properties of the active material. 106 III.2 S t a b i l i t y (and Comparison With Previous Work) The range of pH at which the 8-glucosidase i s stable i s suggested by the method development c a r r i e d out on the FPLC Mono Q anion exchange column. Chromatographic runs carried out i n pH 6.6 and pH 7.6 buffers yielded inactive material implying that the enzyme i s only stable between these pH values. This r e s u l t i s i n reasonable agreement with that of Han and S r i n i v a s a n 5 7 who reported that the enzyme i s stable i n sodium acetate and sodium phosphate buffers between pH 6.5 and pH 7.8. The heat s t a b i l i t y of the enzyme i s hinted at by the Arrhenius p l o t s . The data measured at 40°C lay on the l i n e a r portion of the p l o t . The data measured at 45°C deviated from l i n e a r i t y with a lower rate, F i g . 34. This suggests that the enzyme i s either being denatured or i s less active (but s t i l l stable) at 45°C than at 40°C. Although Han and S r i n i v a s a n 5 7 noted the d i s c o n t i n u i t y i n the Arrhenius plot at 45°C they claimed that the. enzyme was not denatured u n t i l temperatures above 55°C were reached. However, a subsequent study published by Srinivasan and Bumm58 reported that the 8-glucosidase was r a p i d l y inactivated above 45°C. Enzyme which had been dialysed against EDTA to remove any enzyme-bound divalent metal ions (p-galactosidase from E. c o l i requires magnesium ion for complete a c t i v i t y 9 3 ) was found to have i d e n t i c a l k i n e t i c para-meters, with PNPG as substrate, i n the presence and absence of magnesium ion. The Km (PNPG) was i d e n t i c a l , within experimental error, to that 107 measured with untreated enzyme. These r e s u l t s imply that either no divalent metal ion co-factor i s necessary for c a t a l y s i s , or that any divalent cation co-factor i s too t i g h t l y bound to be removed by EDTA. The treated enzyme was found to be i n a c t i v a t e d by i r o n ( I I ) , manganese(II), mercury(II), copper(II) and z i n c ( I I ) ions, but not by calcium ions. When untreated enzyme was used, a c t i v i t y was observed i n the presence of manganese(II) and z i n c ( I I ) ions (the measurement was not made i n the presence of other i o n s ) . A tentative explanation for the increased s t a b i l i t y of the untreated enzyme to i n a c t i v a t i o n by metal ions could involve a non-catalytic, s t r u c t u r a l role for a metal i o n . Han and S r i n i v a s a n 5 7 found that the enzyme was completely i n a c t i -vated by mercury, copper and iron divalent metal ions only. This i n a c t i -vation may have been non-specific (eg. by i n t e r a c t i o n with non-catalytic sulphydryl groups). In t h i s case some protection might be given by the addition of BSA as t h i s would lower the e f f e c t i v e concentration of metal ions reaching the B-glucosidase; some metal ions would bind n o n - s p e c i f i -c a l l y to the BSA. Further work needs to be c a r r i e d out i n order to c l a r i f y the s e n s i t i v i t y of t h i s enzyme to divalent metal ions. In any event, protection from most of these metal ions was afforded by working i n sodium phosphate buffer as most of the metal ion phosphates are almost completely insoluble i n aqueous s o l u t i o n . The B-glucosidase was found to be r e l a t i v e l y stable to storage i n solution (under toluene vapour to i n h i b i t microbial growth); the Km 108 measured (PNPG) using f r e s h l y prepared enzyme was i d e n t i c a l to that measured using enzyme which had been stored i n solution for two months, and which retained 83% of i t s o r i g i n a l a c t i v i t y . I t should be noted that d i l u t e p-glucosidase solutions were inactivated by exposure to surfaces unless BSA was added to increase the protein concentration. L y o p h i l i s a t i o n i n the presence of myo-inositol gave p-glucosidase that retained 62% of i t s o r i g i n a l a c t i v i t y on r e d i s s o l u t i o n . L y o p h i l i s a t i o n may therefore be of some use i n long term storage of the enzyme. Han and S r i n i v a s a n 5 7 reported that myo-inositol completely protected the enzyme a c t i v i t y against l y o p h i l i s a t i o n . This difference may be due to the impure p-glucosidase used by Han and S r i n i v a s a n 5 7 ; contaminating proteins may afford some protection, analogous to the protection against surfaces afforded by BSA. Freezing as a method of storage was not investigated although the c e l l free extract and whole c e l l s could be frozen with only a minimal loss of p-glucosidase a c t i v i t y . 109 III.3 Molecular Weight (and Comparison with Previous Work) The sub-unit molecular weight of the 8-glucosidase determined by SDS-PAGE i s 51,500 Daltons. The molecular weight determined under native conditions using a Superose gel chromatography column i s 75,000 Daltons. This l a t t e r value i s probably low. The support material of the Superose column i s based on the polysaccharide agarose. It i s possible that the 8-glucosidase i s retarded on the column by s p e c i f i c i n t e r a c t i o n s between the (oligosaccharide binding) active s i t e and the polysaccharide backbone of the column packing material. Support for t h i s hypothesis i s the low apparent molecular weight of phosphorylase b as determined by Superose gel chromatography. Phosphorylase b binds oligosaccharides and i s known to ex i s t as a dimer (mol. wt. 194,000) and tetramer (mol. wt. 380,000) i n solution; i t s apparent molecular weight i s 100,000 Daltons. Han and S r i n i v a s a n 5 7 reported a molecular weight for the 8-gluco-sidase of 160,000 Daltons by gel f i l t r a t i o n and 120,000 Daltons by sucrose density gradient c e n t r i f u g a t i o n . However i n both these cases only two standards were used for c a l i b r a t i o n , i . e . the c a l i b r a t i o n curve was the best straight l i n e drawn between two points! In order to c l a r i f y the above re s u l t s the molecular weight should be determined by another technique or using a gel chromatography column with a non polysaccharide support (e.g., one based on polyacrylamide). This work i s being undertaken. It should be noted that i f the enzyme i s an oligomeric protein, 110 then the observation of B-glucosidase a c t i v i t y i n the denaturing SDS-PAGE gel would suggest that the dissociated sub-units r e t a i n some enzymatic a c t i v i t y . I l l III.4 Anomeric Configuration of I n i t i a l Products The anomeric configuration of the i n i t i a l products was determined as described (II.4.4). The r e s u l t s are shown i n F i g . 33. It may be seen that the change i n rotation observed on addition of sodium carbonate suggests that the i n i t i a l product has the 8 configuration. The B-glucosidase i s therefore a r e t a i n i n g B-glucosidase. 15 Time (min.) Figure 33. S p e c i f i c Optical Rotational Changes During Hydrolysis by B-glucosidase. (•), a-D-glucose; (*), 8-D-glucose; (•), B-D-glucopyranosyl f l u o r i d e alone; (x) 8-D-glucopyranosyl f l u o r i d e and 8-glucosidase from A. f a e c a l i s . The measured rotations were converted into s p e c i f i c rotations by using the i n i t i a l weight of glucose or B-D-glucopyranosyl f l u o r i d e . The r o t a t i o n of the sugars was measured at ~35°C and of the f l u o r i d e (with and without B-glucosidase) at ~17°C to minimise non-enzymic hy d r o l y s i s . The arrows mark the time at which sodium carbonate was added. 112 III.5 K i n e t i c and Thermodynamic Data II.5.1 Results The k i n e t i c parameters of the substrates and i n h i b i t o r s studied are shown i n Table X and XI. Arrhenius plots are shown i n F i g . 34. Table X: Ki n e t i c Parameters of B-Glucosidase with Several  Substrates at 37°C and pH 6.8. Substrates Km Vmd Enzyme6 Substrate Vm/Kmf A G ° 8 (mM) (nmoles/minute) cone. cone.(mM) (kJ/mole) PNPG a , b 0.083±0.006 (10.00+0.16) 40.56 0.98-0.016 120 24.23 PNPGal b 2.9±0.5 12.410.7 43.49 40-1.4 4.3 15.07 PNPMan 0.020±0.002 0.0074310.00016 121.1 1.7-0.005 0.37 27.90 PY-G° 4.9 ± 1.2 0.1710.03 43.49 2.5-0.25 0.035 13.72 cellobiose 0.702±0.061 9.6010.38 3.307 2.7-0.13 13.6 18.73 c e l l o t r i o s e 0.387±0.014 8.1110.10 10.19 4.1-0.06 21.1 20.27 cellotetraose 0.332±0.012 7.5210.08 10.19 3.5-0.35 22.7 20.66 cellopentaose 0.312±0.008 6.7310.05 10.19 2.3-0.043 21.6 20.82 lactose 73.216.0 7.4810.48 30.69 32-1.1 0.102 6.74 sucrose 3.82±0.80 1.4910.14 3.114 10-1.3 0.390 14.36 s a l i c i n 0.382±0.025 3.3010.07 3.114 3.3-0.85 8.64 20.30 gentiobiose 3.85±0.68 0.7110.06 29.36 10-0.74 0.18 14.34 g l u c o s y l 3 azide 3.4010.33 0.4910.02 118.9 10-0.81 0.14 14.66 g l u c o s y l 3 f l u o r i d e 3.6910.19 13.010.4 219.0 5.1-0.16 3.52 14.45 2,4-DNPG 0.03110.002 11.110.3 10.25 0.17-0.0097 360 26.77 2,4-TDNPG3 3.6910.19 7.7810.63 32.92 3.3-0.11 2.11 14.45 113 Substrate i n h i b i t i o n was observed with these compounds and obviously i n h i b i t e d points were neglected i n the c a l c u l a t i o n of Km and Vm. These points are c i r c l e d i n the Lineweaver-Burke plots (Appendix 3). k The Km determined for PNPG was 0.083 mM; however, subsequent determinations (during i n h i b i t i o n studies) yielded a Km of ~0.075 mM. This may be due to the use of older enzyme or due to experimental e r r o r . c The large error i n Km i s due to the low substrate concentrations necessitated by the high background absorbance. ^ Vm i s given r e l a t i v e to that amount of enzyme necessary to hydrolyse 10 nmoles of PNPG i n one minute. e The enzyme concentration i s given i n terms of nmoles PNPG hydrolysed i n one minute (at saturating PNPG, Vm). f The units of Vm/Km are min. - 1 L - 1 x 1 0 - 6 . g AG°was calculated from the equation AG°= -RTlnKm. Table XI; K i n e t i c Parameters of 8-Glucosidase with Several  Inh i b i t o r s at 37°C and PH 6.8. Inhibitor Ki R d Type Substrate I n h i b i t o r AG 4 8 (mM) cone. (mM) cone. (mM) (kJ/mole) p-nitrophenyl-8-D-c e l l o b i o s i d e 0.029 0.999 competitive 1.3-0.090 0-0.42 26.94 B-D-glucosylamine 0.40 0.999 competitive 1.3-0.090 0-3.6 20.18 glucono-6-lactone 0.0017 0.999 competitive 1.1-0.075 0-0.013 34.25 a-D-glucopyranose 6.4 (8=0.44) p a r t i a l l y non-competitive 1.1-0.075 0-27 13.03 p-nitrophenyl—a-D-glucopyranoside b — c 1.0-0.045 0-15 — AG0was determined from the equation AG"= -RTlnKi. No k i n e t i c parameters were determined for th i s i n h i b i t o r . The type of i n h i b i t i o n i s discussed i n the text. R i s the c o r r e l a t i o n c o e f f i c i e n t of the re p l o t . Figure 34. Arrhenius plot of InVm and ln(Vm/Km) against 1/T. Lineweaver-Burke plots of data shown i n i n s e t . 115 III.5.2 Comparison with Previous Work The value of Km obtained for PNPG i s somewhat lower than that obtained by Han and S r i n i v a s a n 5 7 of 0.125 mM. This may be because t h e i r k i n e t i c studies were ca r r i e d out on enzyme which had only been p u r i f i e d 50 to 80 f o l d . Han and S r i n i v a s a n 5 7 only investigated c e l l o b i o s e as an i n h i b i t o r of PNPG hyd r o l y s i s . The value of Ki that they found from an approximate determination (a single i n h i b i t o r concentration and several substrate concentrations) of 1 mM i s i n reasonable agreement with the Km of 0.702 mM determined i n t h i s i n v e s t i g a t i o n . An approximate Ki deter-mination was also c a r r i e d out and yielded a comparable value of ~1 mM. The value of Ki determined by Han and S r i n i v a s a n 5 7 for glucose of 1 mM i s i n poor agreement with the value of 6.4 mM determined i n t h i s study. This i s no doubt because an approximate determination assumes that any i n h i b i t i o n i s competitive i n nature. The a c t i v a t i o n energy determined by Han and S r i n i v a s a n 5 7 of 39.7 kJ/mole i s i n reasonable agreement with that calculated from a plot of InVm against r e c i p r o c a l temperature (37.9 kJ/mole, c o e f f i c i e n t of corre-l a t i o n = 0.999). It i s possible that Han and S r i n i v a s a n 5 7 did not consider the v a r i a t i o n of PNP e x t i n c t i o n c o e f f i c i e n t with temperature and t h i s may be the cause of the s l i g h t differences i n a c t i v a t i o n energy c a l c u l a t e d . Of 42 6-glucosidases i s o l a t e d from microbial sources whose a c t i v i t y against PNPG has been measured 1* 9 4 only 3 have smaller Km's 116 than that measured with B-glucosidase from A. f a e c a l i s , and of 39 whose a c t i v i t y against cellobiose has been measured 9 4, only 4 have smaller Km's than that measured with p-glucosidase from A. f a e c a l i s . The e f f i c i e n c y of the p-glucosidase from A. f a e c a l i s , as determined by Km, i s therefore one of the highest measured for a b a c t e r i a l p-glucosidase; b a c t e r i a l p-glucosidases tend to have higher Km's (weaker binding) than those i s o l a t e d from fungal sources 3. This tight binding increases the poten-t i a l u t i l i t y of this enzyme for co-cloning into a c e l l u l a s e complex. The enzyme was found to be the most active (as defined by Vm/Km) in the c e l l o d e x t r i n series (G2-G5) against c e l l o t r i o s e . It i s not unusual for p-glucosidases associated with c e l l u l a s e complexes to have higher a c t i v i t y against higher oligomers of glucose than c e l l o b i o s e 1 > 9 5 • III.5.3 Substrates; Implications for the Active Site and Mechanism A number of deductions may be made about the active s i t e binding s p e c i f i c i t y and mode of action from the k i n e t i c parameters determined. F i r s t of a l l i t i s necessary to confirm that the enzyme i s a ' t y p i c a l ' p-glucosidase, i . e . a ret a i n i n g p-glucosidase having an exo-glucanase a c t i v i t y and a c a t a l y t i c mechanism s i m i l a r to that postulated i n Chapter I ( F i g . 3). Evidence that the enzyme i s a retainin g p-glucosidase has already been discussed ( I I I . 4 ) . Evidence for exo-glucanase a c t i v i t y was obtained during the attempt to measure the k i n e t i c parameters of p-nitrophenyl-p-D-cello-bioside as a substrate. The i n i t i a l reaction rate (as measured by 117 release of PNP) was found to Increase with time and to be higher at low substrate concentrations, F i g . 35. At f i r s t t h i s observation was thought to arise from high substrate i n h i b i t i o n . However product analysis Time (min.) Figure 35. I n i t i a l Rate of Hydrolysis of p-Nitrophenyl -8-D-Cellobioside at a Number of Concentrations. showed that only glucose (and not cellobiose) was produced and t h i s suggested the following explanation for the observed k i n e t i c s . The enzyme cleaves single units from the non-reducing end of the substrate only, I.e. glucose i s cleaved from the PNP-cellobioside to give PNPG and glucose. The concentration of PNPG i s i n i t i a l l y low (below Km PNPG). As the reaction proceeds more PNPG i s produced and as i t s concentration 118 approaches and exceeds Km (PNPG) i t i s better able to compete with PNP-c e l l o b i o s i d e as a substrate and hence the rate of PNP release increases. This enzyme therefore has exo-glucanase a c t i v i t y ( e x c l u s i v e l y ) . The k i n e t i c parameters of PNP-cellobioside as an i n h i b i t o r of the hydrolysis of PNPG were measured and the r e s u l t s are discussed l a t e r . The glycone binding s i t e appears to be able to tolerate a range of sugar moieties, v i z . g l u c o s y l , g a l a c t o s y l , mannosyl (and possibly f r u c t o s y l ) , however the s p e c i f i c i t y of the enzyme, as defined by Vm/Km, i s at least 30 times higher for glucose than any other sugar. The galactosyl moiety i s bound somewhat more weakly than the glucosyl moiety; the difference i n binding energy i s 12 kJ/mole for the pair c e l l o b i o s e / l a c t o s e and 9 kJ/mole for the pair PNPG/PNPGal. This i s close to the binding energy expected from one hydrogen b o n d 1 0 1 . This suggests that the inversion of the 4-hydroxyl either removes one hydrogen bonding i n t e r a c t i o n or introduces a negative i n t e r a c t i o n of the same energy. The enzyme's a b i l i t y to t o l e r a t e galactose may be related to the fact that the 8-glucosidase a c t i v i t y i s induced by addition of lactose to the b a c t e r i a l growth medium. The aglycone binding s i t e has poor s p e c i f i c i t y and can tolerate a wide v a r i e t y of 'aglycones' and linkages v i z . a r y l (PNP, DNP, TDNP, PY, s a l i c y l ) ; inorganic (azide, f l u o r i d e ) ; 8-D-(l,6) (gentiobiose); a-D-(l-l)-B(sucrose, i . e . g l u c o s y l - o c - ( l - l ) - 8 - f r u c t o s i d e ) . This poor s p e c i f i c i t y i s not uncommon95 i n c e l l u l o l y t i c 8-glucosidases; an enzyme which has evolved to degrade c e l l u l o s e for use as a food source does not need the s p e c i f i c i t y of a metabolic enzyme. 119 The 8-glucosidase was, however, found to be completely i n a c t i v e to p-nitrophenyl-a-D-glucopyranoside as a substrate. Glucosidases are normally very s p e c i f i c towards the anomeric configuration of substrates and the a c t i v i t y observed against sucrose here, and by other B-gluco-sidases elsewhere 9 5, may be due to binding of fructose into the glucoside (glycone) s i t e to give a f r u c t o s y l - 8 - ( l - l ) - a - g l u c o s i d e configuration at the active s i t e . The increase i n binding energy on increasing the chain length from one glucose to two (8-D-glucopyranosyl f l u o r i d e or azide to c e l l o b i o s e ) i s 4.2 kJ/mole, and a further increase i n chain length to c e l l o t r i o s e gives r i s e to a further increase i n binding energy of 1.5 kJ/mole. Subsequent increases i n chain length ( c e l l o t e t r a o s e and cellopentaose) do not s i g n i f i c a n t l y increase the strength of binding. This evidence suggests that there are three binding s i t e s with some a f f i n i t y for glucose and that the active s i t e i s arranged such that any a d d i t i o n a l moieties do not s t e r i c a l l y hinder binding to the enzyme. The active s i t e might be envisaged as a tunnel or angled c l e f t , three glucose sub-sites i n length (with hydrolysis taking place between the f i r s t and second sub-sites) and from which any a d d i t i o n a l glucose moieties protrude out from the enzyme surface into s o l u t i o n , F i g . 36. 120 Figure 36. Schematic Representation of 8-Glucosidase Binding S i t e . Gentiobiose binds no more t i g h t l y than a substrate having only one glucose moiety. This suggests that the change from a 8-1-4 linkage to a 8-1-6 linkage either completely destroys the enzyme's a b i l i t y to bind the second glucose moiety or, that a negative i n t e r a c t i o n of equivalent energy to any p o s i t i v e interactions i s Introduced. PY-G also binds no more t i g h t l y than substrates having a small aglycone ( f l u o r i d e or azide) and th i s again suggests either that the 'aglycone' s i t e cannot bind the pyridinium r i n g or, that any p o s i t i v e 121 i n t e r a c t i o n s are counteracted by equivalent negative interactions (possibly between the p o s i t i v e charge on the substrate and a group i n the active s i t e as discussed i n III.5.4). S i m i l a r l y 2,4-TDNPG has a binding energy 12.3 kJ/mole less than 2,4-DNPG, and i n f a c t , not s i g n i f c a n t l y greater than that of the azide or f l u o r i d e . The longer C-S bond length and larger atomic radius of the sulphur must d i s t o r t the structure s u f f i c i e n t l y to either completely destroy the binding interactions between the thiophenolic moiety and the active s i t e , or introduce negative interactions that completely n e u t r a l i s e them. If glucose i s replaced by PNP the binding energy i s increased by 5.5 kJ/mole in the case of ce l l o b i o s e and 6.7 kJ/mole i n the case of c e l l o t r i o s e (from Ki of PNP-cellobioside). The observation that replace-ment of a glucose moiety with a phenolic moiety increases the binding energy hints that the binding interactions might be predominantly hydro-phobic i n nature, although one fortu i t o u s a d d i t i o n a l hydrogen bond could give r i s e to this increase i n binding e n e r g y 1 0 1 ( i t i s also possible that the t i g h t e r binding i s occurring f o r t u i t o u s l y at another s i t e ) . A si m i l a r structure for the second and t h i r d binding s i t e s might be postulated on the basis of the s i m i l a r i t y i n binding energy difference between glucose and PNP found at the two s i t e s . I t may be seen from the range of substrates that the enzyme can catalyse the h e t e r o l y t i c cleavage of C-0, C-N, C-S and C-F bonds. Bond lengths i n model compounds a r e 9 6 > 9 7 : H 3C-(OCH 3) 2, 1.42A; H3C-F, 1.385A; 122 H3C-N, 1.47A; HgC-SH, 1.82A; (pBrCgH^-) 2-S, 1.75A. This shows that the enzyme can tolerate appreciable changes i n an important molecular dimension of the substrate ( i n common with other g l y c o s i d a s e s 1 7 * 9 0 ) . This implies that the acid c a t a l y t i c groups have some degree of freedom movement (or that c a t a l y s i s does not have to involve d i r e c t i n t e r a c t i o n with the exocyclic hetero atom to be cleaved; PY-G cannot undergo acid catalysed h y d r o l y s i s 3 4 ) . The evidence discussed so far suggests a binding s i t e as described ( F i g . 36) i n which the two 'aglycone' s i t e s have some s p e c i f i c i t y for glucose, possibly, by predominantly hydrophobic i n t e r a c t i o n s . It i s h e l p f u l i f the discussion of the mechanism of glycoside hydrolysis (and the evidence for i t ) by B-glucosidase from A. Faecalis c a r r i e d out with reference to following h y p o t h e t i c a l . k i n e t i c mechanism, F i g . 37, and hypothetical chemical mechanism, F i g . 38. Where K 2 represents a non-covalent conformational change (postulated i n the mechanism of some glycosidases, e . g . 1 8 ) , K 3 represents bond cleavage ( F i g . 38) and k+4 represents enzyme deglucosylation. Figure 37. Hypothetical B-Glucosidase Kinetic Mechanism. 123 Figure 38. Hypothetical 6-Glucosidase Chemical Mechanism. The Vm measured for a l l glucosyl substrates does not exceed 10-11 nmoles/min. (glucosyl f l u o r i d e f a l l s into t h i s range i f the possible systematic error i s taken into account; see II.4.2). If bond cleavage (k +^) were rate l i m i t i n g then some c o r r e l a t i o n between Vm and leaving group a b i l i t y , as defined by pKa, would be expected 1 7* 1 8 » 3 4 - 3 6 . PNPG, ce l l o b i o s e , c e l l o t r i o s e , c e l l o t e t r a o s e , B-D-glucopyranosyl f l u o r i d e , 2,4-DNPG and 2,4-TDNPG a l l have comparable values of Vm. As the leaving groups have considerably d i f f e r e n t pKa's the rate l i m i t i n g step cannot be bond cleavage but must be some other step, e.g. (k+4) deglucosylation, (K 2) a slow protein conformational change, release of aglycone etc. Further evidence that bond breaking i s not rate l i m i t i n g comes from the values obtained from the Arrhenius plots for the hydrolysis of PNPG, F i g . 34. A plot of In Vm (representing rate l i m i t i n g step; see 124 Appendix 1) against r e c i p r o c a l temperature yielded an a c t i v a t i o n energy of 37.9 kJ/mole ( c o r r e l a t i o n c o e f f i c i e n t = 0.999). A plot of ln(Vm/Km) (representing f i r s t i r r e v e r s i b l e step; see Appendix 1) against r e c i p r o c a l temperature yielded an a c t i v a t i o n energy of 29.6 kj/mole ( c o r r e l a t i o n c o e f f i c i e n t = 0.990). As the a c t i v a t i o n energy determined for the f i r s t i r r e v e r s i b l e step (presumably bond h e t e r o l y s i s ) i s d i f f e r e n t to that determined for the rate l i m i t i n g step, bond cleavage cannot be rate l i m i t i n g . It i s therefore postulated that for most substrates of th i s enzyme, including the natural substrate, some other step than bond cleavage i s , at least p a r t i a l l y , rate l i m i t i n g . The a c t i v a t i o n energy determined for bond cleavage i n the enzymatically catalysed hydrolysis of PNPG i s considerably less than that reported for the non-enzymic, acid catalysed reaction, 127 kJ/mole" and this shows the high c a t a l y t i c power of this enzyme. The behaviour of the substrate PNPMan i s unusual and deserves some comment. The only difference between this substrate and PNPG i s the a x i a l p o s i t i o n of the 2-hydroxyl. 8-Glucosidases are known to be se n s i t i v e to changes at the 2 p o s i t i o n of the glucose m o i e t y 2 3 * 2 5 , and a review of the l i t e r a t u r e has revealed no other glucosidases which have a c t i v i t y against mannosyl substrates. In the case of B-glucosidase from A. f a e c a l i s PNPMan has an increased binding energy over PNPG of 3.7 kJ/mole and a maximal rate 1/1350 that of PNPG. An approximate i n h i b i t i o n constant was determined (Ki = 0.03) to confirm that hydrolysis was 125 taking place at the same s i t e as PNPG hydro l y s i s . A p p l i c a t i o n of Occam's razor to these observations might give r i s e to the following explanation. The a x i a l 2-hydroxyl may i n t e r a c t with the c a t a l y t i c group -BH ( F i g . 38) thereby both increasing binding interactions and also decreasing c a t a l y t i c e f f i c i e n c y (by i n t e r f e r i n g with acid c a t a l y s i s ) . The s i m i l a r i t y i n Vm observed for the substrates 2,4-DNPG and 2,4-TDNPG deserves some explanation. The acid catalysed rate of hydro-l y s i s of phenyl-B-D-glucopyranoside i s 300 times that of 1-thiophenyl-8-D-glucopyranoside". The reason c i t e d for t h i s " i s that the thio d e r i v a t i v e i s a weaker conjugate acid than the oxygen analogue and i s consequently more r e s i s t a n t to protonation prior to bond h e t e r o l y s i s . In a study involving the enzymically catalysed hydrolysis of these compounds 2 3 the thio analogue was hydrolysed about 6000 times more slowly, and this i s probably because both substrates have to undergo acid hydro-l y s i s i n the enzyme active s i t e . Thiophenoxide ion i s , however, a better leaving group (pKa = 6.61 1 0 0) than phenoxide ion (pKa = 9 . 9 4 1 0 0 ) . If the 2,4-DNP ion, whose pKa = 4.09 1 0 0, i s that much poorer a leaving group than the 2,4-TDNP ion, then neither substrate w i l l require acid c a t a l y t i c assistance for hydrolysis and the thio derivative should undergo bond cleavage at a greater rate than the oxygen d e r i v a t i v e . However, since i t has been shown that bond cleavage i s not rate l i m i t i n g for 2,4-DNPG, the observed rate w i l l depend on the rate l i m i t i n g step not involving bond cleavage. Another, less l i k e l y , explanation i s that while the enzymic rate of C-S bond cleavage might be considerably slower than C-0 bond cleavage, 126 the enzymic rate l i m i t i n g step i s slower than both and thus no difference i s observed. III.5.k I n h i b i t o r s ; Implications f o r the Active Site and Mechanism Inhibitors are extremely useful i n probing both the mechanism and active s i t e environment of B-glucosidases 1 5. The tight binding of the i n h i b i t o r glucono-6-lactone (19.5 kJ/mole more binding energy than the azide or f l u o r i d e ) , whose preferred half chair conformation makes i t an analogue for the glucosyl oxo-carbonium ion, suggests that the t r a n s i t i o n state i s a s t a b i l i s e d oxo-carbonium ion (possibly i n equilibrium with a covalent glycosyl-enzyme species as discussed i n 1.2). 8-Glucosidases have been divided into two groups with regard to the binding of c a t i o n i c and basic substrates and i n h i b i t o r s 1 5 . The f i r s t group binds c a t i o n i c g l y c o s y l derivatives with much higher a f f i n i t y than the i r neutral counterparts. The po s i t i v e charge may be either permanent or can be formed by proton transfer from the enzyme active s i t e . Enzymes from the second group have a high a f f i n i t y only for basic glycosyl d e r i -vatives; permanently c a t i o n i c ones bind s i m i l a r l y to the i r uncharged analogues. This behaviour i n the second group has been p o s t u l a t e d 1 5 to be due to the presence of a c a t i o n i c group located near the putative cata-l y t i c carboxylate (marked -BH i n F i g . 38). This c a t i o n i c group i s proposed to neutra l i s e the influence of the carboxylate group on the 127 permanently c a t i o n l c i n h i b i t o r . Proton donation to the basic glucosyl For both groups the observed e f f e c t s can be taken as evidence for an active s i t e that i s strongly shielded from the aqueous environment since e l e c t r o s t a t i c interactions are very weak i n water. The 8-glucosidase from A. f a e c a l i s can be thought of as belonging to the second group. The permanently c a t i o n i c pyridinium de r i v a t i v e (PY-G) binds no more t i g h t l y than substrates having no charge (azide and f l u o r i d e ) whereas the basic i n h i b i t o r 8-D-glucosylamine has an add i t i o n a l binding energy of 5.7 kJ/mole. anomeric mixture; however, during the 'retention/inversion' study i t became apparent that i t was predominantly i n the oc-configuration. The ki n e t i c s observed were best explained as p a r t i a l l y competitive, F i g . 39. One possible explanation i s that the 8-glucose binds into the t h i r d glucose s i t e and while PNPG can s t i l l bind into the f i r s t two s i t e s , release of products i s hindered by the i n h i b i t i n g a-glucose, F i g . 40. derivatives could be from this c a t i o n i c group or from a neutral a c i d . The glucose used for the i n h i b i t i o n studies was supposed to be an K + + Figure 39. Ki n e t i c Mechanism for P a r t i a l l y Non-Competitive I n h i b i t i o n . 128 Figure 40. Schematic Model for P a r t i a l l y Non-Competitive I n h i b i t i o n . The i n h i b i t i o n observed with p-nitrophenyl-a-D-glucopyranoside was non-linear (see Appendix 3). A k i n e t i c mechanism which f i t s these data i s one involving equivalent, non-interacting s i t e s only one of which can be (competitively) i n h i b i t e d at one t i m e . 9 2 A physical picture of this behaviour i s not obvious. The s l i g h t , but r e a l trend of decreasing Vm with increasing glucose oligomer chain length i s also not r e a d i l y interpreted. The Ki's observed for substrate i n h i b i t i o n by PNPGal and PNPG, of 160 mM and 60 mM respectively, are high enough to have no mechanistic s i g n i f i c a n c e and are probably due to incorrect (unproductive) binding of a second substrate molecule (ESS). 129 II.6 Conclusion Summing up, the active s i t e may have the following features. An angled, hydrophobic c l e f t with three glucose sub-sites from which longer oligomers can protrude into s o l u t i o n . The l a s t two sub-sites are probably s i m i l a r and the binding i n t e r a c t i o n s i n these l a t t e r two s i t e s may be predominantly hydrophobic i n nature. The t r a n s i t i o n state i s probably a s t a b i l i s e d oxocarbonium ion (possibly in equilibrium with a covalent intermediate as discussed i n 1.2). The c a t a l y t i c groups might be arranged as shown i n F i g . 38 and the rate l i m i t i n g step for most substrates i s one which does not involve g l y c o s i d i c bond cleavage. 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(104) A.Cornish-Bowden i n " P r i n c i p l e s of Enzyme K i n e t i c s " , Butterworths (London), 1976. 137 APPENDIX 1 Basic Enzyme Kin e t i c s V 138 The Briggs-Haldane Study State Assumption The following process i s assumed: k k E + S v ^ E S —2->-E + P k - l where E i s free enzyme; S i s free substrate; ES i s enzyme-substrate complex and P i s product(s). (The reverse reaction, E + P >• ES, may be neglected as the concentration of P i s n e g l i g i b l e at the s t a r t of the reaction) At steady state: d[ES] = dt .-. k ^ E H S ] = (k_ x + k )[ES] k L [ S ] Rearranging: [ES] = + k ) [E] (I) -1 p I n i t i a l V e l o c i t y , v i s given by: 139 v = k p[ES] (II) The t o t a l enzyme concentration, [ E l t Is given by: [ E ] t = [E] + [ES] ( I l l ) Dividing II by III and sub s t i t u t i n g the righ t hand side of I for [ES] (k , + k ) A E_ k [E] k t [ S ] [ E ] + (k , + k ) ™ - 1 p Cancelling [E] and s u b s t i t u t i n g Vm (maximal i n i t i a l v e l o c i t y ) for k ^ [ E ] t ; k.[S] (k - + k ) v _ -1 p Vm k [S] 1 + k - l + k P Grouping the rate constants as Km, the Michaelis constant: 140 k . + k where Km = - 1 P_ k l IV i s the Michaelis-Menten equation and Km i s a pseudoequilibrium constant. If a rapid preequilibrium i s assumed, k^ » k _ ^ » k^, then the same equation may be derived, however, Km, i s a true equilibrium ( d i s s o c i a t i o n ) k - l constant: Km = k l Equation IV, as given, i s non-linear, however inversion gives: I - S J L - L . + 1. ( v ) v Vm [S] Vm v ' A plot of ^  vs. -—jy gives a straight l i n e . The ^ - intercept gives ~ and the —r- intercept — . This i s the Lineweaver-Burke plot (see Appendix 3 [ S J Km for examples). I n h i b i t i o n Competitive i n h i b i t i o n arises when an i n h i b i t o r competes for the same s i t e as the substrate. Thus, the rate at saturating substrate (Vm) i s unaffected but the amount of substrate necessary to saturate the s i t e 141 (Km) i n the presence of i n h i b i t o r i s greater. Equation IV becomes: v = M i l [S] + Km (1 + JgL) where [I] i s the i n h i b i t o r concentration and Ki i s the d i s s o c i a t i o n constant for the process: E + 1^^—EI K i I t may be shown that: A replot of K^pp against [I] w i l l be l i n e a r and the [I] intercept w i l l give -Ki (see Appendix 3 for examples). Another plot commonly used to determine Ki i s that of Dixon. ^ i s plotted against [ I ] . The point at which the l i n e s for various values of [S] int e r s e c t gives a value of -Ki on the [I] axis. I f Vm i s known only one value of [I] need be used i n order to calculate an approximate value of K i . The treatment of k i n e t i c data obtained when p a r t i a l l y Km app where Km app i s the apparent Km. 142 non-compe1111ve inhibit ion takes place is non-tr iv ia l and has already been referenced 9 1 . Substrate inhibit ion occurs when a second substrate molecule binds to the ES complex to produce an inactive ES.S complex. Equation IV becomes: v M i l ( V I ) Km + [S] + [S] ^ v ' s i where K . is the inhibit ion constant (dissociation constant) for the s i binding of the second substrate molecule. Inversion of VI gives: 1 Km v Vm[S] Vm K , ' s i at high substrate concentrations the f i r s t term becomes negligible and i t may be shown that a plot of ^ against [S] (at high [S]) w i l l extrapolate to -K on the [S] axis, (see Appendix 3 for examples). More detailed S I discussions of enzyme kinetics may be found in references 103 and 104. The Significance of Vm and Vm/Km Vm is clearly related to the slowest forward step on the enzyme catalysed pathway (k^ in the scheme given). The significance of Vm/Km is less clear intu i t ive ly , however i t may be demonstrated to be related to the f i r s t irreversible step on the catalytic pathway 1 0 2 . This demonstration is non-tr iv ia l and w i l l not be given here. 143 APPENDIX 2 Program for Apple II Computer Used to Determine Km and Vm 144 10 HOME 15 GOSDB 2500 20 PRINT "THIS PROGRAM PROVIDES ESTIMATES" 30 PRINT "OF KM AND VMAX FROM A WEIGHTED" 40 PRINT "LINEAR REGRESSION ANALYSIS. 50 DIM S(10) ,V(10) ,S$(10),V$(10),IY(10),IX(10) 60 D$ = CHR$ (4) + CHR$ (13) 70 K l - 0:FG - 0:FH = 0 80 PRINT : PRINT 90 PRINT "1) Kl DETERMINATION" 100 PRINT : PRINT "2) KM DETERMINATION" 110 PRINT 120 GET Q$ 130 IF ASC (Q$) •» 49 THEN K l •= 1: GOTO 160 140 IF ASC (OJ) •= 50 THEN 160 150 GOTO 120 160 HOME ISO - 0 170 PRINT "SAMPLE TAB( 11)"SUBSTRATE"; TAB ( 23)"RATE"; TAB( 30)"CALC ULATED" 180 PRINT T AB ( 13)"C0NC"; TAB ( 32)"RATE" 190 POKE 34,3 200 HOME 210 N = 0:FG = 0:FH = 0 220 IF K l = 1 THEN IT$ = "INHIBITOR CONC: ":L « 7: IL = 3:FL = 3: GOSUB 1000:IN* = B* 230 PRINT 240 GOSUB 900 245 IF N > 10 THEN 260 250 IF VAL (B$) < > 0 THEN 240 255 IF N < 3 THEN 240 260 GOSUB 1370 280 IT$ •= "ADD A POINT ? (Y/N) " 290 GOSUB 1540 300 IF Q$ » "Y" THEN GOSUB 1440: GOSUB 900 305 IT* " "CONVERT DATA ?(Y/N) ": GOSUB 1540 307 IF Q$ = "Y" THEN FG •= 1: GOSUB 2000: GOSUB 1440 308 IT* » "CALCULATE VM/KM? ": GOSUB 1540 309 IF Q$ - "N" THEN SO = 1: GOTO 840 310 A - 0:B « 0:C = 0:D = 0:E •= 0 320 FOR Z - 1 TO N 330 X - V(Z) k 2 340 Y - X / S(Z) 350 A = A + (V(Z) * X) 360 B - B + X i 2 370 C = C + (Y * V(Z)) 380 D «= D + (X * Y) 390 E = E + Y k 2 400 NEXT Z 410 DE - ((A * E) - (D * C)) 420 KM - ((B * C) - (A * D)) / DE 430 VM » ((B * E) - (D * D)) / DE 440 A - 0:B - 0:C = 0:D - 0:E - 0:F - 0:G - 0:F1 = 0 450 FOR IC - 1 TO N 460 F - VM * S(IC) / (S(IC) + KM) 470 FI - - VM * S(IC) / ((S(IC) + KM) k 2) 480 A - A + F k 2 490 B - B + FI k 2 500 C - C + (F * FI) 145 500 510 520 530 540 550 DE 560 B l 570 B2 580 VR 590 KR 600 S2 610 EK 620 EV 630 IL 640 IL 650 IL 660 IL 670 680 C - C + (F * F l ) D - D + (V(IC) * F) E - E + (V(IC) * F l ) G - G + V(IC) k 2 NEXT IC (A * B) - (C * C) ((B * D) - (C * E)) ((A * E) - (C * D) ) Bl * VM KM + (B2 SQR ((G Bl * S2 * - 3:N0 DE DE S2 / VM * 3:FL 2:FL - 3:N0 3:FL - 3:NO 2:FL - 3:NO GOSUB 1500 FOR Z = 1 TO N Bl) Bl * D SQR (A SQR (B KR ! EK: VR: EV: - B2 * / DE) / DE) GOSUB GOSUB GOSUB GOSUB E) / (N - 2)) 1250:KM$ 1250:EK$ 1250:VM* 1250:EV$ - B$ - B* •= B$ 710 720 730 735 736 737 738 740 750 760 770 775 780 790 800 810 820 840 845 850 855 860 880 / (KR + S(Z)) VC: GOSUB 1250: PRINT ;B* 0:L = 1: GOSUB 1000:SL 690 VC - VR * S(Z) 700 VTAB (Z + 3) HTAB (32) IL » 3:FL - 3:N0 NEXT Z GOSUB 1370 IF VAL (B$) < > 0 THEN 310 IT* » "REFERENCE? (Y/N) ": GOSUB 1540 IF Q$ " "Y" THEN FH «• 1: GOSUB 2200 IT* - "PRINT RESULTS (Y/N) ": GOSUB 1540 IF Q$ - "N" THEN 840 IT* •= "PRINTER SLOT # " : IL = 1:FL <B*) PR# SL HOME PRINT ;"SAMPLE 3"; TAB( 11)"SUBSTRATE"; TAB( 22);"RATE" PRINT TAB( 13)"C0NC" GOSUB 1450 GOSUB 1500 PR# 0 IT* - "GRAPH MENU? (Y/N) "i GOSUB 1540 IF Q* • "t" THEN GOTO 3000 IT$ - "ANOTHER SET OF DATA? "z GOSUB 1540 IF Q$ » "Y" THEN 200 END IL - 1:FL » 0:L - l i l T f - "PRINTER SLOT # ' < B » ) 900 N - N + 1 910 VTAB (N + 3): PRINT TAB( 3);N 920 IT$ - "ENTER RATE : " i L - 7:FL - 3:IL » 3; 930 IF VAL (B$) = 0 THEN VTAB (N + 3): HTAB (3): RETURN 940 VTAB (N + 3): HTAB (22): PRINT ;B| 950 V(N) - VAL (B$):V$(N) - B$ 960 IT$ - "SUBSTRATE CONC: ":L 970 S(N) - VAL (B$):S$(N) = B$ GOSUB 1000:SL GOSUB PRINT i 1000 " ":N 7:IL - 3:FL - 3: GOSUB 1000 980 VTAB (N 990 RETURN 1000 Hi - " + 3): HTAB (11): PRINT ;B$ 146 1000 M* - " " 1010 s* - " 1020 B* - MID$ (M|,1,L) 1030 POKE 35,24: VTAB (23): HTAB (1) 1035 PRINT S* 1037 VTAB (23): HTAB ( l ) 1040 PRINT ; I T | i 1050 1 = 1 1060 VTAB (23): HTAB (20): PRINT S| 1065 HTAB (20): VTAB (23) 1070 PRINT MIDI (B|, LEN (B|) - L + 1, LEN (B|)) 1080 VTAB (23) 1090 HTAB (20 + L) 1100 GET X* 1110 IF X| - CHR* (13) THEN 1220 1120 IF X* < > CHR* (8) THEN 1170 1130 IF I « 1 THEN 1060 1140 B* = MID* ( B * , l , LEN (B*) - 1) 1150 1 = 1 - 1 1160 GOTO 1060 1170 IF I = L + 1 THEN 1060 1180 IF ASC (X*) < 46 OR ASC (X*) > 57 THEN 1060 1190 B* o B* + X* 1200 1 = 1 + 1 1210 GOTO 1060 1220 B* = MID* (B*, LEN (B*) - 1 + 2 , LEN (B*)) 1230 B* = MID* (S*,1,L - LEN (B*)) + B* 1235 RETURN 1240 NO = VAL (B*) 1250 S = SGN (NO) 1260 N2 = 10 k FL 1270 Nl - ABS (NO) + .5 / N2 1280 IP - INT (Nl) 1290 FP - INT (N2 * (Nl - IP)) + N2 1300 B* = STR* (IP):L = LEN (B*):B* = RIGHT* (B*,L) 1310 IF IL = 0 AND IP - 0 THEN B* = "":L = 0 1320 IF S < 0 THEN L = L + 1:B* = "-" + B*: IF IP = 0 AND IL = 1 THEN B* - "-":L = 1 1330 IF IL > L THEN B* = " " + B*:L = L + 1: GOTO 1330 1340 IF FL > 0 THEN B* = B* + "." + RIGHT* ( STR* (FP),FL) 1350 POKE 35,22 1360 RETURN 1370 IT* = "DELETE POINT # " i L = 2: GOSUB 1000:P = VAL (B*) 1380 IF P = 0 THEN RETURN 1390 N = N - 1 1400 FOR Z = P TO N 1410 V(Z) = V(Z + 1):V*(Z) = V*(Z + 1) 1420 S(Z) = S(Z + 1):S*(Z) = S*(Z + 1) 1430 NEXT 1440 HOME 1450 FOR Z = 1 TO N 1460 VTAB (Z + 3): PRINT TAB( 3);Z; 1470 PRINT TAB( 11);S*(Z); TAB( 22);V*(Z) 1480 NEXT 1490 RETURN 1500 VTAB (N + 5): HTAB (2): PRINT "KM - ";KM* j " +/- ";EK * 147 1500 1510 1515 1520 1522 1525 1530 1540 1550 1560 1565 1570 1580 1620 2000 2001 2002 2005 2010 2020 2025 2030 2040 2045 2050 2060 2065 2070 2060 2090 2095 2100 2110 2200 2205 2210 2220 2225 2230 2240 2245 2250 2260 2270 2280 2285 2290 2300 2302 2305 2307 2308 2310 2320 2330 2335 2340 2500 2510 2515 2520 VTAB (N + VTAB (N + IF FG - 1 IF KI » 1 IF KI - 1 5): HTAB (2)t 7): HTAB (2): THEN PRINT " THEN VTAB (N AND FH - 1 THEN E VM=":VE;" NMOLES/MIN." IF KI - 0 AND FH - 1 THEN VM-";VE;" NMOLES/MIN" RETURN POKE 35,24 VTAB (23): HTAB (1) PRINT IT*; VTAB (23): HTAB (20) VTAB (23): HTAB (21) IF Q* = "Y" OR Q* -GOTO 1550 PRINT "KM • PRINT "VM • NMOLES/MIN + 9) : HTAB VTAB (N • " ; KM*;" +/- ";EK* • ";VM*;" +/- ";EV*; II (2): PRINT "[INHIBITOR] - "jIN* + 11): HTAB (2): PRINT "REFERENC VTAB (N + 9): HTAB (2): PRINT "REFERENCE : PRINT S* : GET Q* "N" THEN POKE 35,22i RETURN DATA TREATMENT SUBROUTINE REM IT* - "NEW VALUES?(Y/N) ": GOSUB 1540 IF Q* " "N" GOTO 2080 L - 6 IT* - "E.COEFF.? " + " ( " + EB* + " ) " : IF VAL (B*) - 0 THEN GOTO 2030 EB = VAL (B*):EB* = STR* (EB) IT* = "CELL VOLUME? " + " ( " + VL* + ' IF VAL (B*) - 0 THEN GOTO 2050 VL = VAL (B*):VL* - STR* (VL) IT* » "PATH LENGTH? " + " ( " + LE* + ' IF VAL (B*) » 0 THEN GOTO 2070 LE - VAL (B*):LE* «= STR* (LE) REM TREAT DATA FOR K • 1 TO N V(K) - (V(K) * VL * 1E6) / (EB * LE) V*(K) = STR* (V(K)) NEXT RETURN REM REFERENCE SUBROUTINE L - 6 IT* = "E.CO.REF.?" + " ( " + ES* + " ) " : IF VAL (B*) - 0 THEN GOTO 2230 ES - VAL (B*):ES* - STR* (ES) IT* - "REF. KM? " + " ( " + KT* + ")' GOSUB 1000 GOSUB 1000 GOSUB 1000 GOSUB 1000 GOSUB 1000 VAL (B*) - 0 THEN GOTO 2250 VAL (B*):KT* - STR* (KT) • "CELL VOLUME? " + " ( " + VOS* + " ) " : GOSUB 1000 I GOSUB 1000 " ( " + SO* + GOTO 2305 R* " ( " + VA* + GOTO 2310 ')": GOSUB 1000 GOSUB 1000 IF KT IT* IF VAL (B*) - 0 THEN GOTO 2270 IT* - "[STOCK]? " + " ( " + ST* + ") IF VAL (B*) - 0 THEN GOTO 2290 ST - VAL (B*):ST* - STR* (ST) IT* - "STOCK VOL.? " + IF VAL (B*) - 0 THEN SO - VAL (B*):SO* - ST  * (SO) IT* - "INIT. RATE? " + IF VAL (B*) «= 0 THEN VA - VAL (B*):VA* - STR* (VA) SV - (ST * SO) / VOS VT - (VA * (SV + KT)) / SV VE » VT * VOS * 1E6 / ES GOSUB 1520 RETURN REM SET VARIABLES TO AVOID DIVISION BY ZERO EB » 6220:EB* - "6220":VL - 1.05:VL* = "1.05":LE - 1:LE* - "1":ES 7280:ES* - "7280":KT - ,066:KT* - ".066": VOS - 3.05:VOS* - "3.05"«ST - 2.724:ST* - "2.724":SO - 1.5:S0* - ' 5":VA - .07:VA* - ".07" RETURN 148 GOTO 3200 GOTO 3400 / S(K) - IY(K) - IX(K) GOSUB 1540 3000 REM GRAPHPLOT SUBROUTINE 3010 POKE 34,0: POKE 35,24: HOME :XMAX » - 1E8:YMAX - - 1E8 3020 PRINT "GRAPHPLOT ROUTINE" 3030 PRINT : PRINT 3040 PRINT "1) L/B PLOT" 3050 PRINT : PRINT "2) HILL PLOT 3060 PRINT 3070 GET Q$ 3080 IF ASC (Q$) - 49 THEN 3090 IF ASC (Q$) - 50 THEN 3100 GOTO 3070 3200 REM L/B PLOT 3210 PRINT "L/B PLOT" 3220 PRINT " " 3230 FOR K = 1 TO N 3240 IY(K) = 1 / V(K):IX(K) - 1 3250 IF YMAX < IY(K) THEN YMAX 3260 IF XMAX < IX(K) THEN XMAX 3270 NEXT 3280 PRINT "XMAX=";XMAX: PRINT 3290 PRINT "YMAX"";YMAX 3300 GOSUB 3600 3310 IT| - "REPLOT SAME DATA?"i 3320 IF Q$ - "Y" THEN GOTO 840 3340 GOTO 160 3400 REM HILL PLOT 3410 PRINT "HILL PLOT" 3420 PRINT " " 3421 IF SO - 0 THEN GOTO 3430 3422 IT$ •= "VM?": GOSUB 1000 3425 IF VAL (B$) - 0 THEN GOTO 3422 3427 VR - VAL (B$) 3430 FOR K - 1 TO N 3440 IY(K) - LOG (V(K) / (VR - V(K))):IX(K) - LOG (S(K)) 3450 IF YMAX < IY(K) THEN YMAX - IY(K) 3460 IF XMAX < IX(K) THEN XMAX - IX(K) 3470 NEXT 3475 VTAB (10): HTAB (1) 3480 PRINT "XMAX=";XMAX: PRINT 3490 PRINT "YMAX=";YMAX 3500 GOSUB 3600 3510 IT* - "REPLOT SAME DATA?": GOSUB 1540 3520 IF Q$ «• "Y" THEN GOTO 840 3530 GOTO 160 3600 REM FILE ROUTINE 3610 SI - 2 * N 3620 D$ = CHR$ (4) 3625 PRINT 3630 INPUT "FILE NAME ?";NA$ 3640 PRINT D$;"OPEN";NA| 3650 PRINT D$;"WRITE";NA$ 3660 PRINT SI 3670 FOR K - 1 TO N 3680 PRINT IX(K): PRINT IY(K) 3690 NEXT 3700 PRINT D$;"CLOSE";NA$ 3710 RETURN 149 APPENDIX 3 Graphical Representation of K i n e t i c Data 150 Figure 42: DNPG. 151 F igure 44: 8 -D -G lucopyranosy l a z i d e . 152 Figure 46: PNPGal. 153 Figure 48: PY-G. 154 Figure 50: Sucrose. 155 Figure 52: Lactose. 156 Figure 54: C e l l o t r i o s e . 157 Figure 55: Cellotetraose. Figure 56: Cellopentaose. 158 2 . Lineweaver-Burke Plots f o r Inhibitors. Replots shown in i n s e t s . I n h i b i t o r concentrations shown i n t i t l e s . Figure 57; .20 | .15-O E .10-^ 9 5 -y • ^ . — • I I I ' •10 0 10 20 p-Nitrophenyl-a-D-glucopyranoside. (•), No i n h i b i t o r ; (+), 14.56 mM; (X), 9.860 mM; (•), 7.043 mM (•); 4 .930 mM. .3H Figure 58: p-Nitrophenyl-8-D-cellobioside. (•), No i n h i b i t o r ; (+), 0.4222 mM; (*), 0.1971 mM; ( f ) , 0.08447 mM; (•), 0.01408 mM. 159 Figure 59: 8-D-Glucosylamine. (•), No i n h i b i t o r ; (+), 3.560 mM; (X), 2.494 mM; (+), 1.780 mM; (•), 0.7125 mM. 0 4 .0 8.0 12.0 Vs(mM) Figure 60: D-Glucono - 6-lactone. (•), No i n h i b i t o r ; (+), 12.29 \M; (*), 6.147 pM; («)), 3.192 pM; (•), 1.596 uM. 160 Figure 62: a-D-Glucopyranose. Secondary r e p l o t s . 161 3 . Substrate Inhibition; Reciprocal Plots. 0 40 s (mw) 162 Addendum Molecular Weight Determination on Waters 1-250 HPLC Gel column Molecular weight was determined on a Waters S i l i c a based HPLC gel column ( i n order to avoid s p e c i f i c i n t e r a c t i o n s ) . Table XII: Molecular Weight Markers for 1-250 column. Protein Molecular weight (Daltons) Log (Molecular weight) Retn. (ml) F e r r i t i n Phosphorylase Aldolase Black Albumin BSA Ovalbumin Lactalbumin S-Glucosidase from A. f a e c a l i s 450,000 198,000 158,000 68,000 66,000 45,000 1 4 , 5 0 0 98,000 (Determined) 5.653 6.73 5.288 7.07 5.199 7.66 4.833 9.25 4.820 9.22 4.653 9.41 4.161 1 0 . 7 8 8.55 163 Figure 65: Plot of Log (Molecular Weight) Against Rf f 1-250 Gel Chromatography. The enzyme i s therefore a dimer of subunit molecular weight 50, Daltons. 

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