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Stabilization and mechanistic studies of soluble processing alpha-glucosidase I from saccharomyces cerevisiae Li, Patrick Ying Xia 1999

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S T A B I L I Z A T I O N A N D M E C H A N I S T I C STUDIES OF S O L U B L E PROCESSING A L P H A - G L U C O S L D A S E I F R O M SACCHAROMYCES CEREVISIAE by P A T R I C K Y I N G X I A L I B.Sc(Honours), The Memorial University of Newfoundland, 1996 A THESIS S U B M I T T E D FN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R O F S C I E N C E in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Food Science We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF BRITISH C O L U M B I A October 1999 © Patrick Ying Xia Li, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^QOcl S c i € ^ C - f ? The University of British Columbia Vancouver, Canada Date ^fj DE-6 (2/88) Abstract Processing a-glucosidase I (E.C.3.2.1.106) is a key enzyme in the biosynthesis of asparagine-linked oligosaccharides catalyzing the first processing event after the en bloc transfer of Glc3Man9GlcNAc2 to proteins. The stability and mechanism of this enzyme were investigated in this research. The enzyme was isolated from dry yeast cells and was further purified using affinity chromatography with an enzyme inhibitor, N-methyl-N-(5-carboxypentyl)-l-deoxynojirimycin, as the ligand, and Concanavalin A-Sepharose chromatography. To improve the long-term stability of the enzyme, various additives were added into the phosphate buffer used for extraction and isolation. These additives were different protease inhibitors, including protease inhibitor cocktail [Sigma, product No. P8215, containing 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatin A, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane(E-64), and 1,10-phenanthroline] (1%) and phenylmethylsulfonyl fluoride (PMSF) (100 uM); reducing agent - dithiothreitol (DTT) (0.5 mM); chelating agent - ethylenediaminetetraacetic acid (EDTA) (0.2 mg/ml); polyol -glycerol (10%); and protective protein solution - bovine serum albumin (BSA) (1 mg/ml). The stability of the isolated enzyme was studied when kept at 4°C, -25°C, -80°C, and after freeze drying. Kinetic evaluation of several synthesized substrate analogues and chemical modification of the active site of the enzyme were also attempted. It was showed in this research that processing a-glucosidase I could be further purified using Concanavalin A -Sepharose chromatography. The stability of the enzyme could be much improved by the addition of glycerol, EDTA, BSA, PMSF and protease inhibitors during isolation. Extremely low freezing temperature (-25°C and -80°C) could help to retain enzyme activity during storage. Processing a-glucosidase I was an inverting enzyme which catalyzes ii hydrolysis with inversion of anomeric configuration. Synthetic substrate analogues 2'Fluoro-aGlc(l-2)aGlc-0-grease and 2'N3-aGlc(l-2)aGlc-0-grease were competitive inhibitors of the enzyme and at least one acidic amino acid might be present at the active site of the enzyme. It is believed that results from this research will provide useful information in the design of appropriate enzyme inhibitors that could be beneficial to human health. iii Table of Contents ABSTRACT ii LIST OF TABLES ' vii LIST OF FIGURES viii A C K N O W L E D G E M E N T xi 1. INTRODUCTION 1 2. L ITERATURE REVIEW 6 2.1. Purification of Glucosidase 1 6 2.1.1. Isolation of Glucosidase 1 6 2.1.1.1. Category of Enzymes and General Procedure of Isolating an Enzyme 6 2.1.1.2. Glucosidases and the Isolation of Glucosidase 1 9 2.1.1.3. Characteristics of Glucosidase 1 15 2.1.2. Stabilization of Glucosidase 1 15 2.2. Mechanistic Studies of Glucosidase 1 17 2.2.1. Stereochemistry of Glucosidase 1 17 2.2.1.1. Stereochemistry 17 2.2.1.2. N M R Spectroscopy 21 2.2.2. Catalytic Amino Acid Identification at the Active Site 23 2.2.3. Inhibitors of Glucosidase 1 25 3. METHODS A N D MATERIALS 32 3.1 Materials 32 3.2 Purification of Soluble Glucosidase 1 36 iv 3.2.1. Isolation of Soluble Glucosidase 1 36 3.2.2. Assay of Glucosidase I Activity 36 3.2.3. Protein Assay 37 3.2.4. Stabilization of Glucosidase 1 38 3.2.5. Highly Purification of Glucosidase I with Affinity Chromatography 41 3.2.5.1. Affinity Chromatography with an Enzyme Inhibitor astheLigand 41 3.2.5.2. Concanavalin A-Sepharose Chromatography 42 3.3. Mechanistic Studies of Glucosidase I 44 3.3.1. Stereochemistry of Glucosidase I Reaction 44 3.3.2. Kinetic Evaluation of Glucosidase I Substrate Analogues 44 3.3.3. Site-Specific Chemical Modification of Glucosidase 1 45 3.3.3.1. Inhibitory Effect of E D A C on Glucosidase 1 45 3.3.3.2. Test for Active Site Components of Glucosidase 1 46 RESULTS A N D DISCUSSION 50 4.1. Isolation of Glucosidase 1 50 4.2. Stabilization of Glucosidase 1 53 4.3. Further Purification of Glucosidase I with Affinity Chromatography 61 4.3.1. Affinity Chromatography with N-methyl-N-(5-carboxypentyl)-1-deoxynojirimycin as the Ligand 61 4.3.2. Concanavalin A-Sepharose Chromatography 62 4.4. Stereochemistry of Glucosidase I Reaction 65 4.5. Kinetic Evaluation of Glucosidase I Substrate Analogues 68 4.6. Site-Specific Chemical Modification of Glucosidase 1 75 5. CONCLUSIONS 82 6. REFERENCES 83 APPENDIX 1 92 vi List of Tables Table Page 1. Cell disintegration techniques (Adapted from Scopes, 1982) 8 2. Structures of substrate analogues 33 3. Combination of additives used to stabilize glucosidase I during isolation and storage 40 4. Isolation of soluble glucosidase I (from 25 g yeast cells) 51 5. The interference of various additive solutions with the glucosidase I assay 54 6. Total activity and % yield of glucosidase I isolated from 25 g of dry yeast cells with different combination additives 55 7. Inhibition of glucosidase I by substrate analogues at concentrations of 1 and 10 mM 69 vii List of Figures Figure Page 1. Structures of the typical N-linked oligosaccharides, (a) The high-mannose, (b) the complex and (c) the hybrid types of oligosaccharides. Boxed is the common core 2 2. The processing pathway for modification of the N-linked oligosaccharides 3 3. Basic principle of affinity chromatography. A ligand L is covalently attached to the backbone matrix. Only proteins P i with a specific affinity for L bind to the absorbent. Proteins P2 pass through unaffected 10 4. Structure of (a) 1-deoxynojirimycin, (b) ^-(S-carboxypentyl)-!-deoxynojirimycin, and (c) A/-methyl-A/-(5-carboxypentyl)-1-deoxynojirimycin.. 12 5. Reaction pathway for glucosidase I activity assay 14 6. Catalytic mechanism for retaining glycosidases 18 7. Catalytic mechanism for inverting glycosidases 19 8. Effect of external magnetic field on a proton, (a) Parallel and antiparallel alignments of the proton spin axis relative to the direction of the field, (b) The proton can occupy one of two quantum states depending on the direction of its alignment. The energy difference AE between the states increases with the magnitude of the applied field. In the presence of a magnetic field, electromagnetic radiation with a frequency satisfying the relation AE = hv is absorbed and induces a transition between the states (Adapted from Porile, 1987) 22 9. The various nuclear magnetic resonance spectral parameters (Adapted from James, 1975) 24 10. Characteristic relation between 1/v and 1/[S] for various types of inhibition 27 11. Mixed-type inhibition 29 12. Site of action of various inhibitors that act on the glucosidases involved in the processing pathway 30 13. Procedure of ED A C inhibitory effect on glucosidase I 47 14. Procedure for the site-specific chemical modification of glucosidase 1 49 Vlll 15. Stability of glucosidase I isolated with various combination of additives (Letters A - G in the legend refer to Combinations A - G used to stabilize glucosidase I during isolation and storage, as Combinations A - G in Table 3)... 56 16. Stability of glucosidase I stored under sterile conditions compared to glucosidase I stored under non-sterile conditions. Enzyme was isolated with Combination D (10 mM phosphate buffer, 10% glycerol and 100 u M PMSF).. . 58 17. Stability of glucosidase I after freeze drying and freezing treatments 59 18. SDS-PAGE pattern of enzyme fraction from Con A-sepharose chromatography. Bands in Lane 1 represent the broad range SDS-PAGE standard, of which molecular weight of each band is showed on the left. Bands in Lane 2 represent the purified enzyme fraction 63 19. Time course of glucosidase I reaction (Palcic et al, 1999) 66 20. Lineweaver Burk inverse plot of 2'N3-aGlc(l-2)aGlc-0-grease inhibition of glucosidase I. The original data was fit by non-linear regression to the Michaelis-Menton equation to obtain K m (apparent) and V m a x at different inhibitor concentrations 70 21. Lineweaver Burk inverse plot of 2'Fluoro-aGlc(l-2)aGlc-0-grease inhibition of glucosidase I. The original data was fit by non-linear regression to the Michaelis-Menton equation to obtain K m (apparent) and V m a x at different inhibitor concentrations 71 22. Replot of apparent K m obtained from non-linear regression analysis of inhibition of glucosidase I at 0, 0.5 and 1.0 mM of 2 'N 3 -aGlc(l-2)aGlc-0-grease (see Fig.20). K; was calculated as the negative x-intercept 72 23. Replot of apparent K m obtained from non-linear regression analysis of inhibition of glucosidase I at 0, 1.0 and 2.5 mM of 2Tluoro-aGlc(l-2)aGlc-0-grease (see Fig.21). Kj was calculated as the negative x-intercept 73 24. Inhibitory effect of E D A C on glucosidase I activity. Enzyme was isolated using buffers containing Combination D additives (10 mM phosphate buffer, 10% glycerol and 100 u M PMSF). Results presented are the average of duplicate assays at each time point. Error bars represent the range for each pair of readings 76 25. Percentage of initial enzyme activity remaining in samples of glucosidase I. Treatment A - dialysis in buffer containing 10 mM phosphate, 10% glycerol and 100 u M PMSF, pH 6.8 (buffer A) overnight; Treatment B - added with 10 mM D N J M and incubated at 4°C for 1 hr; Treatment C - added with 50 mM E D A C and incubated at 4°C for 1 hr after Treatment B; Treatment D -ix dialysis in buffer A overnight after Treatment C; Treatment E - added with 50 mM ED A C and incubated at 4°C for 1 hr after Treatment D. Enzyme was isolated with Combination D additives (buffer A). Results presented are the average of duplicate assays at each time point. Error bars represent the range for each pair of readings 77 26. Percentage of initial enzyme activity remaining in samples of glucosidase I. Treatment A - dialysis in buffer containing 10 mM phosphate, 10% glycerol and 100 u M PMSF, pH 6.8 (buffer A) overnight; Treatment B - added with 50 mM ED AC and incubated at 4°C for 1 hr; Treatment C - dialysis in buffer A overnight after Treatment B. Enzyme was isolated with Combination D additives (buffer A). Results presented are the average of duplicate assays at each time point. Error bars represent the range for each pair of readings 78 27. The reaction of carbodiimide with protein carboxyl groups 80 x A C K N O W L E D G E M E N T I am grateful to Dr. C. Seaman for her constant assistance, patience and encouragement during the years I devoted to this work. I also would like to thank the committee members, Drs. T. Durance, E. Li-Chan, and B. Skura for their numerous helpful suggestions and review of this work. I wish to thank Dr. Om Srivastava at the Alberta Research Council, Edmonton, Alberta for the synthesis of trisaccharide substrate analogues. Finally, I am grateful to Mrs. V. Skura and Mr. S. Yee for their much-needed technical support. xi 1. Introduction Asparagine-linked (or N-linked) glycoproteins are a group of biologically important proteins. In animal cells, most membrane receptors, including those for low density lipoproteins (LDL), for insulin, and for acetylcholine, are asparagine-linked glycoproteins, as are many other biologically important proteins such as lysosomal enzymes, serum proteins and secreted proteins (Elbein, 1991a). The carbohydrate portions of these proteins are assembled via the dolichol cycle in the rough endoplasmic reticulum (ER) (Kornfeld and Kornfeld, 1985). There are three major classes of N-linked oligosaccharides - the high-mannose, the complex, and the hybrid types (Fig. 1), which share a common biosynthetic pathway, and which contain a common core region of: Manoc 1,6(Mana 1,3)ManB 1,4-GlcNAcP 1,4-GlcNAc All of the various N-linked oligosaccharides arise from the common intermediate, Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol, a lipid-linked oligosaccharide that is assembled in the ER of the cell by the sequential addition of the sugars N-acetyl-D-glucosamine, mannose, and glucose to the lipid carrier, dolichol-P (P=pyrophosphate) (Hubbard and Ivatt, 1981). The oligosaccharide is then transferred from this lipid carrier to specific asparagine residues that are in the consensus sequence, Asn-X-Ser(Thr). Once the oligosaccharide is transferred to protein, the oligosaccharide chain begins to undergo a series of processing events (Fig. 2). These events start in the rough ER with the trimming of the glucosyl residues and continue in the Golgi compartment where one or more mannosyl residues are also clipped. Depending on individual glycoproteins, additional glycosylations of the trimmed oligosaccharide, mediated by the Golgi-localized glycosyltransferases, may 1 (a) Manal ^-2Manal SA = sialic acid Man = mannose Gal = galactose Asn = asparagine GlcNAc = N-acetyl-D-glucosamine ^ 6 Manal ! 3 j Manal->2Manal ^ j Manpl->4GlcNAcpl->4GlcNAcA>Asn Manal ^ 2Manal -> 2 Manal ! (b) SA->Gal^GlcNAc^Manal ! X ! Manp 1 ->4GlcNAcp 1 ->4GlcNAc-»Asn i ^ 3 SA^>Gal->GlcNAc->Manal (c) Manal ^ 6 Manal ^ 3 ^ 6 : M a n a l x | Manpi^4GlcNAcpl^4GlcNAc->Asn 1 3 1 SA->Gal->GlcNAc^>Manal ^ ! Figure 1. Structures of the typical N-linked oligosaccharides, (a) The high-mannose, (b) the complex and (c) the hybrid types of oligosaccharides. Boxed is the common core. 2 (Glc) 3(Man) 9(GlcNAc) 2-PP-Dol I Man-Man ^ Man | Man-Man / Man-GlcNAc-GlcNAc-Asn Glc-Glc-Glc-Man-Man-Man 1 Glc < ^ Glucosidase I Glc < ^ Glucosidase II Man-Man ^ Man I Man-Man / Man-GlcNAc-GlcNAc-Asn -> High mannose A T A T ^ I chains Man-Man-Man 1 Mannosidase I Man < | Man ^ Man j Man / Man-GlcNAc-GlcNAc-Asn M a n X I GlcNAc > ^ GlcNAc Transferase I Man x Hybrid ^ X Man x , c h a i n s Man / Man-GlcNAc-GlcNAc-Asn GlcNAc-Man X I Man « | , Mannosidase II Man | Man-GlcNAc-GlcNAc-Asn G l c N A c - M a n ' I I I Man-GlcNAc-GlcNAc-Asn SA-Gal-GlcNAc-Man I SA-Gal-GlcNAc-Man X Figure 2. The processing pathway for modification of the N-linked oligosaccharides. 3 occur to yield the final oligosaccharide moieties in the proteins (Kornfeld and Kornfeld, 1985). As the first processing enzyme, processing a-glucosidase I (glucosidase I) triggers the onset of the above processing events by removing the outermost al,2-linked glucose from the newly constructed glycoprotein. It is therefore an important factor in the overall regulation of glycoprotein biosynthesis. Some researchers are examining its role in promoting protein targeting and folding (Faye et al, 1993). Some researchers are studying its role in the biosynthesis of cell wall components (Abeijon and Chen, 1998; Shahinian et al, 1998). Some glucosidase I inhibitors have been shown to act as antiviral and antitumor agents (Elbein, 1991b; Fenouillet et al, 1994; Fischer et al, 1995; van den Broek et al, 1996). Many viruses are enveloped viruses. In most cases, the virus contains one or more glycoproteins that make up the envelope structure, and these glycoproteins are frequently of the N-linked type (Elbein, 1991b). Therefore, inhibiting the enzyme glucosidase I could affect N-linked glycosylation or block modifications in the processing of the N-linked oligosaccharides, and thus have profound effects on viral growth and replication. As well, N-linked glycoproteins are the components of the tumor cell surface involved in the recognition and lysis. This suggests that N-linked oligosaccharides participate in recognition and/or lysis events. In other words, glucosidase I is an important enzyme in glycoprotein biosynthesis. Inhibitors of this enzyme are potential antiviral and anti-cancer agents. Better understanding of the characteristics of this enzyme will therefore facilitate development of inhibitors which could be used in treatment of some main human diseases including Acquired 4 Immunodeficiency Syndrome (AIDS) and cancers. Investigations of the active site components of glucosidase I have recently started through studies on the structure-function relationships of glucosidase I (Pukazhenthi et al, 1993; Romaniouk and Vijay, 1997). It is believed that this valuable information will provide important direction in the design of appropriate enzyme inhibitors that could be beneficial to human health (Palcic et al, 1999). The objectives of this research project were to improve techniques used to isolate and stabilize glucosidase I; to increase understanding of glucosidase I mechanism by revealing the stereochemistry of glucosidase I reaction, by evaluating the kinetic effect of substrate analogues, and by investigating the active site components of glucosidase I. 5 2. L i t e r a t u r e R e v i e w 2.1. Purification of Glucosidase I 2.1.1. Isolation of Glucosidase I 2.1.1.1. Category of Enzymes and General Procedure of Isolating an Enzyme Enzymes are an important group of proteins. They control the chemical reactions of life through their catalytic action. In general, enzymes can be classified into six categories: 1) oxidoreductases which are involved in biological oxidations and reductions; 2) transferases which catalyze the transfer of a group from one substrate to another; 3) hydrolases that catalyze the formation of two products from a substrate by a hydrolytic reaction; 4) lyases that catalyze the addition of groups to double bonds or their formation; 5) isomerases that catalyze isomerisation; and 6) ligases (or synthetases) which join together two molecules, simultaneously with the breakdown of ATP (Wiseman and Gould, 1971). Hydrolases are further classified into esterases, thiolesterases, phosphatases, glycosidases, and peptidases (Dixon and Webb, 1964). Glycosidases catalyze the hydrolysis of glycosidic bonds. Processing cc-glucosidase I, the enzyme studied in this research work, is within this category. Glycosidases play a central role in a number of biological processes that are of significant interest for biochemistry, medicine, and biotechnology. For example, lysozyme is present in egg white and human tears and cleaves bacterial cell wall polysaccharides, thereby serving as a relatively broad range bactericidal agent (Withers and Aebersold, 1995); also, specific enzymes are increasingly used for food processing (invertase for production of "invert" sugar, cellulases for fruit juice processing, p-galactosidase for lactose reduction) (Coughlan and Hazlewood, 1993). Enzymes are found in nature in complex mixtures, usually in cells that contain a 6 hundred or more different enzymes, and in order to study an enzyme properly it must be isolated. The serious isolation of enzymes can be dated back to 1920s (Dixon and Webb, 1964). The first crystalline enzyme (urease) was obtained by Sumner (1926). By 1940 about twenty highly purified enzymes had been obtained, and this process has continued at an ever-increasing rate. In the isolation of an enzyme, the general procedure usually includes: 1) determining a convenient quantitative test of activity by which the enzyme can be estimated. Since a large part of the time spent in enzyme isolation is used up in testing the activity of the different fractions, it is most desirable that the test method should be a rapid one; 2) searching for a rich source of the enzyme. The amount of an enzyme may be very different in different sources and by choosing a source which is rich in the enzyme not only is more enzyme obtained, but the degree of purification required to obtain the pure enzyme is much less, perhaps by a factor of 10 or even 100; 3) making an extract containing the enzyme from the chosen raw material. There are many methods to disintegrate cells because there are many different types of cell. Most cells have particular characteristics that need special attention during disintegration. Animal tissues vary from the very easily broken erythrocytes to tough collagenous material such as blood vessels and other smooth-muscle-containing tissue. Plant cells are generally more difficult to disrupt than animal cells because of the cellulosic cell walls. Bacteria vary from fairly fragile organisms that can be broken up by digestive enzymes or osmotic shock to more resilient species with thick cell walls, needing vigorous mechanical treatment for disintegration. Table 1 gives a list of techniques that can be used. These techniques have been illustrated in great detail (Scopes, 1982); 4) separating the enzyme from numerous other substances. The enzyme is usually separated by precipitation first. This can be done by change of pH, by heating, by salts, by selective 7 Table 1. Cell disintegration techniques (Adapted from Scopes, 1982). Technique Gentle Cell lysis Enzyme digestion Chemical solubilization / autolysis Hand homogenizer Mincing (grinding) Moderate Blade homogenizer (Waring-type) Grinding with abrasive (e.g., sand, alumina) Example Erythrocytes Lysozyme treatment of bacteria Toluene extraction of yeast Liver tissue Muscle etc. Muscle tissue, most animal tissues, plant tissues Plant tissues, bacteria Principle Osmotic disruption of cell membrane Cell wall digested, leading to osmotic disruption of cell membrane Cell wall (membrane) partially solubilized chemically; lytic enzymes that are released complete the process Cells forced through narrow gap, rips off cell membrane Cells disrupted during mincing process by shear force Chopping action breaks up large cells, shears apart smaller ones Microroughness rips off cell walls Vigorous French press Ultrasonication Bead mill Manton-Gaulin homogenizer Bacteria, plant cells Cell suspensions Cell suspensions Cell suspensions Cells forced through small orifice at very high pressure; shear forces disrupt cells Micro-scale high-pressure sound waves cause disruption by shear forces and cavitation Rapid vibration with glass beads rips cell walls off As for French press above, but on a larger scale denaturation, with organic solvents, or with organic polymers. Then the enzyme can be further purified by adsorption and other more advanced techniques such as chromatography, gel filtration, electrophoresis, isoelectric focusing, liquid phase partitioning, and ultrafiltration, etc. Among these techniques, affinity chromatography is by far the most useful and successfully applied method for providing high purity proteins. For example, non-affinity methods generally provide less than a 20-fold purification of proteins, but affinity chromatography routinely achieves purification of between 1,000- and 10,000-fold. The basic principle of affinity chromatography is described in Fig.3. 2.1.1.2. Glucosidases and the Isolation of Glucosidase I Glucosidases (E.C.3.2.1) belong to the category of hydrolases. Initial investigations on the processing of newly synthesized glycoproteins indicated the removal of glucose residues prior to mannose from lipid-linked oligosaccharides (Ugalde et al, 1980). Two glucosidase activities were subsequently identified (Chen and Lennarz, 1978; Grinna and Robbins, 1979; Elting etal, 1980) and termed glucosidase I (E.C.3.2.1.106), which removes the terminal cxl,2-linked glucose from GlcsMangGlcNAc^, and glucosidase II, which removes the remaining two a 1,3-linked glucose residues from the precursor. As an important enzyme in the glycoprotein biosynthesis, glucosidase I has gained increasing attention in research. It has been isolated from various sources, including rat liver (Grinna & Robbins, 1979; Ugalde etal, 1980), mung bean seedlings (Szumilo etal, 1986), mammary tissue (Shailubhai et al, 1987; Shailubhai et al, 1991), pig liver (Bause et al, 1989), yeast (Bause et al, 1986; Neverova et al, 1994) and human hippocampus (Kalz-Fuller et al, 1995). Glucosidase I has been obtained in two forms. One form is membrane-bound and its activity was stimulated by detergent, whereas the other form was soluble 9 Figure 3. Basic principle of affinity chromatography. A ligand L is covalently attached to the backbone matrix. Only proteins Pi with a specific affinity for L bind to the absorbent. Proteins P2 pass through unaffected. 10 without detergent. During the isolation steps of glucosidase I in these studies, the raw materials from different sources usually underwent a series of homogenization, filtration and centrifugation in extraction buffer. Then the microsomal glucosidase I (membrane-bound form) was solubilized with detergents such as Triton X-100 and Lubrol PX, or the soluble glucosidase I was precipitated out with ammonium sulfate. Both forms of crude enzyme were then subjected to chromatographic techniques. For example, in isolating microsomal glucosidase I from mung bean seedlings (Szumilo et al, 1986), when the solubilized enzyme fraction was chromatographed on DE-52, glucosidase I activity was detected. The glucosidase I was purified about 200-fold by chromatography on hydroxylapatite, Sephadex G-200, and dextran-Sepharose. In isolating soluble glucosidase I from yeast cells (Neverova et al., 1994), after the yeast cells were broken up by passing through a French press twice and cell debris were removed by centrifugation, the enzyme was isolated from a 20-60% ammonium sulfate protein fraction of the supernatant which was then applied to a Toyopearl D E A E column and a Mono-Q HR 5/5 column. In addition to the isolation steps listed above, glucosidase I has also been highly purified in some studies using affinity adsorption chromatography. There are three types of affinity chromatography. These include the use of specific ligands (e.g., enzyme inhibitors, hormones, neurotransmitters), a general ligand (lectins), and immunoaffinity ligands (antibodies). With various derivatives of a potent glucosidase I inhibitor - 1-deoxynojirimycin as the specific ligand, including N-(5-carboxypentyl)-1-deoxynojirimycin (Hettkamp et al, 1984; Bause et al, 1986; Pukazhenthi et al, 1993) and N-methyl-N-(5-carboxypentyl)- 1-deoxynojirimycin (Bause et al. 1991) (refer to Fig.4 for structures), 11 Figure 4. Structure of (a) 1-deoxynojirimycin, (b) N-(5-carboxypentyI)-l-deoxynojirimycin, and (c) A/-methyl-Ar-(5-carboxypentyl)-l-deoxynojirimycin. 12 glucosidase I was purified from about 200-fold to about 960-fold over the crude microsomal fraction. Since glucosidase I itself is a glycoprotein with high-mannose oligosaccharide(s) (Bause et al, 1986), Concanavalin A affinity chromatography has also been used for further purification of glucosidase I (Bause et al, 1986; Szumilo et al, 1986) and found to be able to purify the enzyme by about 200-fold to 1,900-fold depending on the techniques used in the isolation steps. Concanavalin A is one type of lectin, which are defined as carbohydrate-binding proteins of nonimmune origin, and it offers a rapid method of purifying glycoproteins. At neutral pH concanavalin A exists as a tetramer with each subunit possessing one specific carbohydrate-binding site (Sadhu and Magnuson, 1989). The carbohydrate-binding site is specific for structures containing a-linked mannose or glucose. Two pyranoses, a-D-mannopyranose and a-D-glucopyranose, are recognized for binding by Concanavalin A, and the affinity of Concanavalin A toward a-D-mannopyranose is ca. 3.5-fold higher than toward a-D-glucopyranose (Hassing and Goldstein, 1970). Two main methods have been used to assay the glucosidase I activity in those studies. Initially, glucosidase I activity was monitored by incubating the enzyme preparation with the natural substrate - Glc3Man9GlcNAc2 obtained from microsomes or cell culture. The Glc was randomly labeled using 3 H or 1 4 C. The release of [3H]glucose or [14C]glucose was determined and one unit of enzyme activity was defined as the amount of enzyme capable of releasing 1% [3H]glucose or [14C]glucose from the labeled oligosaccharide per minute. In a recent study (Neverova et al, 1994) a trisaccharide substrate, a-D-Glcl-»2a-D-Glcl^3a-D-Glc-0(CH 2 )8COOCH 3 , was used to determine enzyme activity. The terminal glucose was released from the substrate by the enzyme and quantitated using glucose oxidase, peroxidase, and o-dianisidine (Fig. 5). One unit of enzyme activity is 13 .OH ,1 Ho^ r ^ ^ ° \ o o ^ - J ^ r ^ HO HO O H OH L 4 o 0 H O (CH 2 ) 8 COOMe aGlc(1^2)aGlc(1^3)aGlc-0-R Glucosidase-I O H HO HO O H HO O HO O H Reducing Glc 0 H O (CH 2 ) 8 COOMe + aGlc(l->3) aGlc-O-R 2(H 20),0 2 Glucose Oxidase Gluconic acid + H2O2 o-Dianisidine (colorless) Peroxidase Oxidized o-dianisidine (Brown color) Figure 5. Reaction pathway for glucosidase I activity assay. 14 defined as the amount of enzyme required to release 1 nmol glucose/hr at 37°C. 2.1.1.3. Characteristics of Glucosidase I Based on the enzyme isolated from various sources in these studies, it has been shown that glucosidase I has a pH optimum of 6.2 - 7.0; the enzyme does not require any metal ions for its activity; 1-deoxynojirimycin, a basic analogue of glucose, and its derivatives are potent inhibitors of the enzyme; it is also inhibited by kojibiose and by tris; the enzyme itself is a glycoprotein with high-mannose oligosaccharide; the yeast microsomal enzyme has a subunit with molecular mass of 95 kDa while microsomal enzyme from other sources has a subunit with molecular mass of 85 kDa. The amino acid sequences of glucosidase I from pig liver and human hippocampus were found to have little difference within the coding sequence, demonstrating the close evolutionary relationship between the enzymes from both organisms. 2.1.2. Stabilization of Glucosidase I Enzymes are relatively fragile substances, with a tendency to undergo inactivation under unsuitable conditions. They are usually exposed to three types of factors which may lead to loss of activity: 1) denaturation; 2) inactivation of catalytic site; and 3) proteolysis (Scopes, 1982). Therefore, in the handling of enzymes, the first consideration must always be to avoid inactivation. Denaturation can be minimized if the principal effectors of denaturation are avoided. These are extremes of pH, temperature, and denaturants such as some organic solvents. The natural pH inside a cell would normally be in the range 6-8, so buffers within this pH range, or as close as possible to the actual pH in the tissue concerned, should protect against pH denaturation. The inactivation of enzymes due to a specific effect 15 on the catalytic site is more difficult to avoid. Loss of cofactors can be prevented, if they are known, by adding them back or including them in the buffers used. Proteolysis, which is caused by the presence of proteolytic enzymes, can be minimized by mixing proteolytic inhibitors in the buffers. In purification of glucosidase I, the enzyme was found to be very unstable (Szumilo et al, 1986; Shailubhai et al, 1987). For example, the enzyme lost more than 50% of its activity at room temperature after 24 hours (Shailubhai et al, 1987). Therefore, it is necessary to find a way to stabilize the enzyme in order to further study its characteristics. 16 2.2. Mechanistic Studies of Glucosidase I 2.2.1. Stereochemistry of Glucosidase I 2.2.1.1. Stereochemistry There are two major categories of glycosidases. Those that hydrolyze the glycosidic bond with net inversion of configuration are termed inverting enzymes, and those that do so with net retention of anomeric configuration are termed retaining enzymes (Koshland, 1953). The likely catalytic mechanisms for retaining glycosidases and inverting glycosidases were proposed by Koshland (1953) more than forty years ago and have largely stood the test of time. Retaining glycosidases are generally believed to function through a double displacement mechanism in which a glycosyl-enzyme intermediate is formed and hydrolyzed via oxocarbonium ion-like transition states as illustrated in Fig. 6. The reaction is facilitated by acid^ase catalysis. It is probable that the same group plays both roles. Inverting glycosidases are believed to function by a single-step mechanism in which a water molecule effects a direct displacement of the glycosidic leaving group from the anomeric center as shown in Fig.7. Again this displacement mechanism is general acid/base catalyzed, but with one active site amino acid acting as the general base, helping to deprotonate the nucleophilic water molecule, and the other amino acid acting as a general acid, protonating the departing oxygen atom in a concerted fashion as the bond cleaves. The reaction proceeds via an oxocarbonium ion-like transition state. This model is supported by 3D structures that are available for several different inverting glycosidases such as the cellulases from Trichoderma reesei (Rouvinen et al, 1990) and Thermomonospora fusca (Spezio et al, 1993), soybean B-amylase (Mikami et al, 1993), and Aspergillus awamori glucoamylase 17 Figure 6. Catalytic mechanism for retaining glycosidases. 18 19 (Aleshin et al, 1994). In all cases studied, it is apparent that these acids and bases are the carboxylic side chains of aspartic or glutamic acids. Although glucosidase I has attracted much attention recently, not many mechanistic or structural studies have been carried out on this enzyme, in part due to difficulties in obtaining large quantities of the natural substrate and the enzyme. As a result, no one has yet been able to place glucosidase I into one of the two categories. In order to show that if glucosidase I is an inverting enzyme or a retaining enzyme, it is necessary to study the stereochemical course of glucosidase I reaction. As pointed out by Koshland (1953), an enzymatic reaction can be considered as a reaction between organic molecules in which the catalyst happens to be a protein. Such a consideration allows the tools of physical organic chemistry to be applied to the study of the enzyme mechanism. One of the particularly powerful tools used in the elucidation of chemical mechanisms is stereochemistry. Stereochemical analysis has traditionally provided fundamental information about transition states and intermediates in enzymatic as well as nonenzymatic reactions. In enzymatic reactions, stereochemical analysis generates information about both the chemical reaction mechanisms and specific binding interactions between substrates or inhibitors and enzymatic binding sites (Frey, 1986). This information complements mechanistic information obtained in kinetic, spectroscopic, inhibition, and other studies, bringing the reaction mechanism into clearer focus. In the past two decades, stereochemical course of various enzymatic reactions have been studied and shown to be useful in categorizing enzymes. Among these studies, N M R (Nuclear Magnetic Resonance) spectroscopy was one of the techniques that have been successfully employed. For example, the N M R spectrum led to the conclusion that 20 staphylococcal nuclease is an inverting enzyme (Hibler et al, 1986). 2.2.1.2. NMR Spectroscopy N M R is a spectroscopic technique based on the magnetic properties of atomic nuclei. It is a powerful tool for structural studies. Like electrons, nucleons possess angular momentum as a result of their intrinsic spin and orbital motion. When several nucleons are present in a single nucleus, their angular momenta combine to yield a net angular momentum characteristic of the nucleus as a whole. This angular momentum is known as the nuclear spin. A particle possessing angular momentum is affected by an external magnetic field. For a hydrogen atom, since the nucleus is just a proton, which has spin V2, the application of a magnetic field causes a hydrogen atom to assume one of two different orientations with respect to the field. As shown in Fig. 8, the spin axis may be aligned parallel to the magnetic field direction or antiparallel to it. The atom has a different energy corresponding to each of these alignments, and the application of a magnetic field gives rise to two different quantum states. If electromagnetic radiation with a frequency satisfies the energy difference between these states on a sample placed in a magnetic field, the radiation can be absorbed. The absorption associated with the transition between the two states constitutes the nuclear magnetic resonance. The technique is valuable because the external magnetic field is modified by the presence of electrons in the vicinity of the absorbing hydrogen atom. The magnitude of the field required to induce absorption consequently depends on the specific chemical environment of each hydrogen atom present in a molecule. Therefore, different molecules will have unique N M R spectrum of their own. For a simple compound, peaks appearing in the N M R spectrum are singlets. For a more complicated molecule, hydrogen nuclei can interact with other hydrogen nuclei possessing magnetic 21 Parallel External field Anti-parallel (a) W W 1 h v Magnetic field (b) Figure 8. Effect of external magnetic field on a proton, (a) Parallel and antiparallel alignments of the proton spin axis relative to the direction of the field, (b) The proton can occupy one of two quantum states depending on the direction of its alignment. The energy difference AE between the states increases with the magnitude of the applied field. In the presence of a magnetic field, electromagnetic radiation with a frequency satisfying the relation AE = hv is absorbed and induces a transition between the states (Adapted from Porile, 1987). 22 moments to cause mutual splitting of the nuclear magnetic resonance peaks into multiplets. The coupling of one set of equivalent spins with another set of spins is termed "spin-spin coupling" or "spin-spin splitting". The magnitude of the splitting is designated by the spin-spin coupling constant J and is independent of the applied magnetic field strength. An unperturbed nucleus or equivalent set of nuclei will give a singlet. Multiplet splitting of a resonance from a set of equivalent nuclei will be determined by nearby sets of equivalent nuclei. There are various nuclear magnetic resonance spectral parameters that are summarized in Fig. 9. The N M R spectrum is composed of bell-shaped resonance peaks with the following characteristics: 1) chemical shift (S) - denotes peak position relative to a reference (H0); 2) amplitude - height of peak in arbitrary units; 3) intensity - integrated area under the peak; 4) linewidth ( W i / 2 ) - measure of the width of the peak at half maximal amplitude (in Hz); and 5) spin-spin coupling constant (Jac)- separation (in Hz) between the peaks in a multiplet. These N M R spectrum parameters provide an indication of the types and relative numbers of groups present, and thus provide a detailed picture of molecular structure. By continuously monitoring the changes of the substrate(s) and product(s) using the N M R spectroscopic technique, the stereochemical course of an enzymatic reaction can be then studied. 2.2.2. Catalytic Amino Acid Identification at the Active Site The different stereochemical outcomes of retaining and inverting glycosidases demand quite different catalytic mechanisms, and thus are likely to have different active site components and structures. There are several different approaches for the identification of catalytic amino acid residues at the active site of glycosidases. These include methods using 23 Coupling Constant J a c Coupling Constant J a c i l l ! Linewidth W1/2 Intensity (Area) Reference 1 S b Sa Chemical Shifts H 0 -Figure 9. The various nuclear magnetic resonance spectral parameters (Adapted from James, 1975). 24 sequence alignment, 3D structure analysis, group-specific labels, derivatization with affinity labels, and active site labeling with mechanism-based inhibitors. The pros and cons of these approaches have been reviewed in great detail by Withers and Aebersold (1995). Group-specific labels are designed to form stable, covalent bonds with specific functional groups located at the active site. Various group-specific reagents have been used in this approach, including Woodward's reagent K and ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDAC) for carboxyl groups (Tomme and Claeyssens, 1989; Bray and Clarke, 1994), phenylglyoxal and 2,3-butandione for arginine, N-bromosuccinimide for tryptophan, tetranitromethane for tyrosine, N-ethylmaleimide for cysteine, and diethylpyrocarbonate for histidine (Romaniouk and Vijay, 1997). Due to the simplicity of group-specific labels, scientists have started to look at the active site components of glucosidase I with this approach in the past decade. Using D N J M (1-deoxynojirimycin) to protect the active site and various specific reagents to modify the enzyme, it was found that a cysteine (Pukazhenthi et al, 1993), an arginine and a tryptophan residue (Romaniouk and Vijay, 1997) are located at the active site of glucosidase I from bovine mammary gland. 2.2.3. Inhibitors of Glucosidase I Any substance that decreases the velocity of an enzyme-catalyzed reaction can be considered to be an inhibitor. Enzyme inhibitors are generally classified into three simple categories including competitive inhibitor, noncompetitive inhibitor and uncompetitive inhibitor. A competitive inhibitor is a substance that combines with free enzyme in a manner that prevents substrate binding. The inhibitor and the substrate are mutually exclusive, 25 which is often because of true competition for the same site. The equilibria describing competitive inhibition can be shown as below: K s K p E + S <=± ES — • E + P EI Where K ; = [E][I]/[EI], K s = [E][S]/[ES], and K p = rate constant for the breakdown of ES to E + P. The competition and mutual exclusion of S and I are clearly seen. An increase in [S] at constant [I] decreases the degree of inhibition. An increase in [I] at constant [S] increases the degree of inhibition. The lower the value of Ki , the greater is the degree of inhibition at any given [S] and [I]. Each of the various categories of inhibition has its own characteristic 1/v versus 1/[S] plot (Fig. 10). For competitive inhibition, a competitive inhibitor acts only to increase the apparent K m (measured as [S] required for V2 V m a x ) for the substrate. As [I] increases, K m a p p increases but the Vmax remains unchanged. For noncompetitive inhibition, a classical noncompetitive inhibitor decreases V m a x , but has no effect on the K m value. For uncompetitive inhibition, both K m a p p and V m a x will change as [I] changes. V m a x will be lower in the presence of an uncompetitive inhibitor than the V m a x in the absence of an uncompetitive inhibitor. Unlike noncompetitive inhibition, however, the apparent K m value will decrease. In addition to the above three simple categories of inhibition, there is a more complicated type of inhibition - mixed type inhibition. As the name suggests, a mixed type + I 26 Figure 10. Characteristic relation between 1/v and 1/[S] for various types of inhibition. 27 inhibitor affects both the V m a x and K m values of an enzyme-catalyzed reaction. The general 1/v versus 1/[S] plot for this type of inhibition is shown in Fig.l 1. Inhibitors of glycoprotein processing have been very useful as probes to study the role of N-linked oligosaccharides in glycoprotein function (Elbein, 1991a). Several inhibitors were found to be able to block the modification of N-linked oligosaccharides by inhibiting the glycosidases that remove sugars from the Glc3Man9(Glc-NAc)2-protein (Fig. 12). Among these compounds, the indolizidine alkaloid castanospermine that is found in the seeds of the Australian tree, Castanospermum australe (Szumilo et al., 1986) and the azahexose deoxynojirimycin, found in the culture medium of certain bacteria of the Streptomyces or Bacillus genera (Niwa et al, 1970), were shown to be potent inhibitors of glucosidase I. Castanospermine achieves 50% inhibition at about 2 or 3 u M while deoxynojirimycin inhibits the enzyme activity by 50% at about 5 to 7 uM. In other studies (Shailubhai et al, 1987), heavy-metal ions including Co 2 + , Zn 2 + , A g + and H g 2 + strongly inhibited glucosidase I activity. This type of inhibition can be reversed by adding an excess of dithiothreitol. Effect of a number of mono- and di-saccharides were also tested as potential inhibitors (Shailubhai et al, 1987). Among the saccharides tested, only kojibiose, an cc-l,2-linked glucose disaccharide, inhibited the enzyme significantly, which supports the postulate that the distal glucose residue is attached on oc-l,2-glucosidically to the Glc2Man9GlcNAc2 oligosaccharide core. Both castanospermine and deoxynojirimycin are potent inhibitors of viral replication including the AIDS virus and tumor cell growth in tissue-culture cells (Walker et al, 1988; Montefiori etal, 1988; Goiters etal, 1987). Consequently, several of their derivatives have been tested in clinical trials as possible chemotherapeutic agents (van den Broek et al, 28 Figure 11. Mixed-type inhibition. 29 H0\ ' O H - N Glc 3 Man 9 GlcNAc 2 -PP-Dol Man-Man \ Man HO O H Castanospermine Man-Man / Glc-Glc-Glc-Man-Man-Man \ Man-GlcNAc 2-Protein HO O H Deoxynojirimycin C H 2 O H HO Deoxymannoj irimycin Man-Man / Man-Man-Man Man-GlcNAc2-Protein-> High manno se chains Man \ Man / Man \ Man Man I \ 4 Man Man-GlcNAc 2-Protein Man Man / GlcNAc-Man \ Man-GlcNAc2-Protein-> Hybrid / chains OH , „ OH Swainsonine Man GlcNAc-Man 2 Man Man-GlcNAc 2-Protein I I Complex chains Figure 12. Site of action of various inhibitors that act on the glucosidases involved in the processing pathway. 30 1996). 31 3. Methods and Materials 3.1. Materials Autolyzing yeast cells (Saccharomyces cerevisiae), phenylmethylsulfonyl fluoride (PMSF), p-nitrophenyl-alpha-D-glucopyranoside (PNPG), protease inhibitor cocktail [containing 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatin A, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane(E-64), and 1,10-phenanthroline], 1-deoxynojirimycin (DNJM), Concavalin A, sepharose, a-methylmannoside, oc-methylglucoside, thioglycolate, CAPS transfer buffer [10 mM 3-(cyclohexylamino)-l-propanesulfonic acid], B-mercaptoethanol, bromophenol blue, ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) (obtained from Sigma-Aldrich Canada Ltd, Oakville, ON); monobasic sodium phosphate (NaH 2P0 4), glycerol, ammonium sulfate, Tris, sodium carbonate, dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), MgCl 2 , MnCl 2 , CaCl 2, methanol, ethanol, acrylamide, bisacrylamide, sodium dodecyl sulfate (SDS), glycine, N,N,N',N'-tetramethylethylenediamine (TEMED), cupric acetate (obtained from Fisher Scientific, Fair Lawn, NJ); bovine serum albumin (BSA), ammonium persulfate (APS), coomassie blue, polyvinylidene difluoride (PVDF) protein sequencing membrane, broad range SDS-PAGE standard (obtained from Bio-Rad Laboratories, Richmond, CA); NaCl, acetic acid, isopropanol (obtained from B D H Inc., Toronto, ON); synthesized trisaccharide substrate [a-D-Glcl^2a-D-Glcl^3a-D-Glc-0(CH 2 )8COOCH 3 ] and various substrate analogues (see Table 2) (obtained from Dr. Om Srivastava at the Alberta Research Council, Edmonton, AB). All electrophoresis reagents were electrophoresis grade. All other chemicals used were reagent grade. 32 Table 2. Structures of substrate analogues. Compound Structure cxGlc( 1 -2)aGlc( 1-3 )aGlc-0-grease OH OGr 3 'Deoxy-aGlc( 1 -2)aGlc-0-grease O O G r O H ^ — O H 4' Deoxy-aGlc( 1 -2)aGlc-0-grease O OGr 6'Deoxy-aGlc( 1 -2)aGlc-0-grease 0 OGr 33 2'0-MethylaGlc(l-2)aGlc-0-grease O OGr H O 3 H C O 4' 0-MethylaGlc( 1 -2)aGlc-0-grease O OGr 3HCO 2 'NH 2 -aGlc( 1 -2)aGlc-0-grease ^ O H O OGr H O 2 H N ^ - O H 2'N3-aGlc(l-2)aGlc-0-grease O OGr H O 3 N ^ O H 34 6' Chloro-aGlc( 1 -2)aGlc-0-grease O H 6Chloro-aGlc( 1 -2)aGlc-0-grease O OGr HO O H ^ O H 2 Tluoro-aGlc( 1 -2)aGlc-0-grease O OGr For all the structures above OGr = O OMe 35 3.2. Purification of Soluble Glucosidase I 3.2.1. Isolation of Soluble Glucosidase I The protocol for isolation of soluble glucosidase I was modified from a previous study (Neverova et al, 1994). A 25 g sample of dry yeast cells was suspended in 200 ml of 10 mM phosphate buffer containing 10% glycerol and 100 u M PMSF, pH 6.8 (buffer A), and passed through a French press twice at 12,000 psi. Cell debris was removed by centrifugation at 8,000 x g for 30 min. The supernatant was centrifuged at 200,000 x g for 45 min. Soluble glucosidase I was precipitated out from a 20 - 60% ammonium sulfate fraction of the resulting supernatant. The precipitate was dissolved in 10 ml of buffer A and dialyzed against 2 L of buffer A overnight at 4°C. Dialyzed extract was applied to a Toyopearl D E A E column (2.5 x 20 cm) equilibrated with the same buffer. The column was washed with buffer A containing 0.1 M NaCl. After the protein peak had eluted, glucosidase I was eluted with buffer A containing 0.3 M NaCl. This fraction was dialyzed against 4 L of buffer A at 4°C for 3 hours and then subjected to FPLC with a Source Q column (0.5 x 5 cm). The column was washed with buffer A containing 0.2 M NaCl, and the enzyme was eluted with a linear gradient of 0.2 to 0.5 M NaCl, at a flow rate of 0.20 ml/min with 1-ml fractions collected. Eighty ml of eluant was collected during a typical chromatographic run. Fractions containing glucosidase I were pooled, and stored at 4°C. 3.2.2. Assay of Glucosidase I Activity The glucosidase I enzyme activity was assayed with a synthetic trisaccharide substrate, a-D-Glcl-»2a-D-Glcl->3a-D-Glc-0(CH 2)8COOCH 3 , as described in Neverova et al. (1994). Reactions were initiated by the addition of 4 ul enzyme solution to 1 ul of 10 36 mM a -D-Glc l^2a -D-Glc l^3a -D-Glc -0 (CH 2 ) 8 COOCH 3 in 500-ul microfuge tubes. Tubes were vortexed, briefly microfuged, and incubated at 37°C for 1 hour. The tubes were again briefly microfuged and the reaction was quenched with Tris, using 25 u.1 of 1.25 M Tris-HCl, pH 7.6. The reaction solution was then transferred to a well on a microassay plate and 20 u.1 of buffer A was added, followed by 250 ul of combined color reagent, containing glucose oxidase (5 units/ml), horseradish peroxidase (1 purpurogallin unit/ml) and o-dianisidine dihydrochloride (40 u.g/ml). The solutions were protected from light by covering with aluminum foil and left to develop for 30 min at 37°C. Absorbances were read at 450 nm with a microplate reader and the amount of glucose released was calculated from the glucose standard curve. The reaction scheme for the assay is shown in Fig.5. One unit of activity was defined as the amount of enzyme required for the release of 1 nmol glucose/hr at 37°C. The activity assay was done on collected fractions at each step during isolation and the activity, total activity, and specific activity of each fraction were calculated. Glucosidase II activity and other glucosidase activities were also determined for each fraction collected during the isolation. This was done by incubating 5 ul of 20 mM p-nitrophenyl-alpha-D-glucopyranoside and 15 ul of enzyme fraction in a well on a microassay plate at 37°C for 5 min to 1 hour. Reactions were terminated by the addition of 200 ul of 0.2 M sodium carbonate. The absorbance of each well at 405 nm was read with a microplate reader. The amount of enzyme required to release 1 nmol glucose per hour was used to define the enzyme activity. 3.2.3. Protein Assay The protein content of each fraction collected during isolation steps was determined 37 ' according to the standard instructions of the Bio-Rad Protein Assay (standard procedure for microtiter plates). At first, dye reagent was prepared by diluting 1 part Dye Reagent Concentrate with 4 parts DDI water and filtered through a Whatman #1 filter to remove particulates. Five dilutions of B S A standard were then prepared. Protein solutions were normally assayed in triplicate. Ten microliters of each standard and sample solution was pipetted into separate microtiter plate wells and 200 u.1 of diluted dye reagent was added to each well. Sample and reagent were then mixed thoroughly, clean tips were replaced and reagent was added to the next set of wells. The microtiter plate was incubated at room temperature for 15 minutes. Finally, absorbance was measured at 595 nm with a microplate reader. 3.2.4. Stabilization of Glucosidase I The stability of glucosidase I in solution is very poor, especially in the early stages of the enzyme isolation. To try to improve both the yield and long term stability of the enzyme, various additives were added to the phosphate buffer used for extraction and isolation. These additives are different protease inhibitors, including protease inhibitor cocktail [Sigma, product No. P8215, containing 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatin A, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane(E-64), and 1,10-phenanthroline] (1%) and PMSF (100 um); reducing agent - DTT (0.5 mM); chelating agent - E D T A (0.2 mg/ml); polyol - glycerol (10%); and protective protein solution - B S A (1 mg/ml). The levels of concentrations used were based on studies on various enzymes, especially on different glycosidases. Phosphate buffer was used to control the pH. It can stabilize many enzymes 38 (Scopes, 1982). The protease inhibitor cocktail, PMSF and E D T A were chosen because they could inhibit various proteolytic enzymes present during the isolation steps which would hydrolyze glucosidase I. The other additives were included in the present study because they have been used during the isolation of various enzymes to help retain enzyme activity. DTT prevents the enzyme from being oxidized; glycerol is a protein stabilizer since it can form strong hydrogen bonds with water, effectively lowering the water activity; and B S A can act as an alternative substrate for proteases which may be present (Scopes, 1982). Various combinations of additives used during the isolation of the enzyme are listed in Table 3. At different time intervals over the period of 30 days, the activity of isolated glucosidase I was assayed. The enzyme activity was then compared to the activity of the control (isolated with 10 mM phosphate buffer only). Since it has been mentioned that keeping an enzyme in the deep-freeze state can prolong the half life of the enzyme, or drying an enzyme in high vacuum at low temperature from the frozen state can give an active and soluble powder which can be stored at room temperature (Dixon and Webb, 1964), the effects of freezing (at -25°C and -80°C) and freeze drying treatments on the stability of glucosidase I were also studied in this research. For freeze drying treatment, aliquots (4 ul) of glucosidase I were stored at -25°C after being freeze dried (medium drying rate, Speed Vac, Model SC 110). Enzyme activity was assayed at different time intervals. Each aliquot was rehydrated with 4 ul of distilled water before the activity assay. For freezing treatment, aliquots of 4 ul of glucosidase I were stored in 500 ul microfuge tubes at -25°C and -80°C respectively and then the enzyme activity was assayed at different time intervals. It is possible that proteases arising from contaminating bacteria were responsible for 39 x x X X X X X X X X X X X X PQ X X e o 08 C = o U c3 O o o tu tzi <u -*-» o u O H "<? 0 s (U o C/5 PH O o 40 the loss of glucosidase I activity. Accordingly, the stability of sterile and non-sterile glucosidase I activity over a period of 30 days were also compared. Isolated glucosidase I (using Combination D, i.e. 10 mM phosphate buffer, 10% glycerol and 100 u M PMSF) was sterilized by passing through a 0.22 um sterile Millipore Filter Unit into a sterile 50-ml Falcon tube. Aliquots (4 ul) of sterile glucosidase I were aseptically transferred into microfuge tubes and stored at 4°C. All microfuge tubes and pipette tips used for the transfer were previously autoclaved. Non-sterile glucosidase I from the same isolation batch was stored in non-sterile microfuge tubes at 4°C as well. The activity of both sterile and non-sterile glucosidase I was assayed at different time intervals and compared. 3.2.5. Highly Purification of Glucosidase I with Affinity Chromatography Taking advantage of the high specificity of affinity chromatography, inhibitor ligand affinity chromatography and Con A chromatography were attempted to further purify glucosidase I in this research work in order to study the amino sequence of glucosidase I. 3.2.5.1. Affinity Chromatography with an Enzyme Inhibitor as the Ligand Synthesis of the affinity ligand, N-methyl-N-(5-carboxypentyl)-l-deoxynojirimycin, and the coupling of the affinity ligand to AH-Sepharose 4B were done by a collaborating laboratory, according to the procedure in Bause et al. (1991). After glucosidase I was isolated from yeast cells, as described above, 9.5 ml of the enzyme solution was added to 10 ml of packed gel washed with buffer A in a 50-ml falcon tube. The mixture was tumbled at 4°C overnight. After brief centrifugation, the supernatant was removed and assayed for glucosidase I activity. The gel was decanted into a column and washed with 200 ml of 100 mM phosphate buffer at pH 6.8, followed by 200 ml of 10 41 mM phosphate buffer at pH 6.8. The washed gel was transferred to a 50-ml falcon tube and tumbled overnight at 4°C with 10 ml of 10 mM deoxynojirimycin (DNJM). The supernatant was then concentrated to 50 pi by a 10 K Centricon concentrator (Amicon) and washed twice with 500 ul of phosphate buffer. The final concentrated liquid (purified glucosidase I) was assayed for glucosidase I activity and run on a 10% SDS-PAGE gel. 3.2.5.2. Concanavalin A-Sepharose Chromatography Pooled enzyme fractions after FPLC step were loaded into a 10 ml Concanavalin A-sepharose column which had been equilibrated with buffer A containing 0.1 mM MgCl 2 , 0.1 mM MnCl 2 , 0.1 mM CaCl 2 and 0.5 M NaCl. Then, 10 ml of 1 M a-methylmannoside or a-methylglucoside in buffer A with 0.2 mg/ml E D T A was applied to the column, the column was stoppered and stored at 4°C overnight in contact with the a-methylmannoside or a-methylglucoside. The next day, the elution was continued and additional amounts of a-methylmannoside or a-methylglucoside were passed through the column. The first 40 ml of elution fluid was collected in 40 1-ml fractions and fractions with glucosidase I activity were pooled. The pooled enzyme fractions were then concentrated to 50 ul by a 10 K Centricon concentrator and washed with 500 ul of phosphate buffer twice. The final concentrated liquid (purified glucosidase I) was assayed for glucosidase I activity and run on a 10% SDS-P A G E gel. Sequencing of glucosidase I purified from Con A-Sepharose chromatography was attempted. The purified enzyme fraction was run on a SDS-PAGE gel (with 10% resolving gel). While the enzyme fraction was being electrophoresed, a sheet of polyvinylidene difluoride (PVDF) membrane was wetted with 100% methanol and equilibrated with CAPS transfer buffer [10 mM 3-(cyclohexylamino)-l-propanesulfonic acid, 10% methanol, pH 42 11.0]. After electrophoresis was completed, the gel was assembled into a blot transfer cassette and the enzyme protein was electroblotted for 6 hours at 30 volts. The PVDF membrane was then washed with two changes of CAPS transfer buffer and stained with 0.1% Coomassie blue in 50% methanol for 5 min. The blot was destained with 50% methanol for 10 min. The membrane was then washed with several changes of distilled-deionized water for 5-10 min and air dried. The stained blot was stored dry at -25°C. The band corresponding to glucosidase I was cut from the membrane and excess membrane carefully trimmed away to give a 2x4 mm segment. The segment was sent for amino sequencing analysis at the Nucleic Acid and Protein Service (NAPS) unit of the Biotechnology Laboratory, UBC. 43 3.3. Mechanistic Studies of Glucosidase I 3.3.1. Stereochemistry of Glucosidase I Reaction Glucosidase I was isolated as previously described (using Combination D) and exchanged into 50 mM deuterated sodium phosphate buffer, pD 7.1 (where pD is the uncorrected pH meter reading) using Centricon 10 units (Amicon). The enzyme was then assayed for activity and the stereochemical course of the glucosidase I reaction was studied using H ' - N M R spectroscopy. H ' - N M R spectra were acquired at 27.0°C on a Varian Inova 600 spectrometer using Vr>IMR 5.3B software. Chemical shifts were reported relative to 0.1% external acetone at 2.225 ppm. After initial acquisition of the reference enzyme solution spectrum (700 ul in 5 mm N M R tubes), 60 ul of aliquots of concentrated trisaccharide substrate were added in to give a final concentration of 9.3 mM. A spectrum was recorded after 3 min, then every 5 min for 2.5 hr and finally every 30 min until hydrolysis was complete. In this way the reaction's progress was monitored for up to 15 hr. Spectra were processed with a Gaussian window function of width at/4 and shifted by at/6. A first order baseline correction was applied prior to data analysis, integration and plotting. For integrations, the H 1 signal that remained unchanged over the course of the reaction, was used as a reference. 3.3.2. Kinetic Evaluation of Glucosidase I Substrate Analogues Since inhibition studies of glucosidase I could be beneficial in preventing or curing various human diseases including ADDS and cancer, further exploration of the possible inhibitory mechanism of this enzyme was done by evaluating the kinetic effects of some synthesized glucosidase I substrate analogues. The glucosidase I substrate was cxGlc(l-44 2)aGlc(l-3)aGlc-0-(CH 2 ) 8 COOCH 3 which was first used by Neverova et al. (1994). Substrate analogues including 3'Deoxy-aGlc(l-2)aGlc-0-(CH 2)8COOCH 3, 4'Deoxy-aGlc(l-2)aGlc-0-(CH 2 ) 8 COOCH 3 , 6'Deoxy-aGlc(l-2)aGlc-0-(CH 2) 8COOCH 3 , 2 '0-Methyl-aGlc(l-2)aGlc-0-(CH 2) 8COOCH 3 , 4'0-Methyl-aGlc(l-2)aGlc-0-(CH 2 ) 8 COOCH 3 , 2 'NH 2 -aGlc(l-2)aGlc-0-(CH 2 ) 8 COOCH 3 , 2 'N 3-aGlc(l-2)aGlc-0-(CH 2 ) 8 COOCH 3 , 6'Chloro-aGlc( 1 -2)aGlc-0-(CH 2 ) 8 COOCH 3 , 6Chloro-aGlc( 1 -2)aGlc-0-(CH 2 ) 8 COOCH 3 and 2'Fluoro-aGlc(l-2)aGlc-0-(CH 2) 8COOCH 3 were synthesized by Dr. Om Srivastava at the Alberta Research Council, Edmonton, Alberta. Structures of these substrate analogues can be found in Table 2. Prior to a full kinetic evaluation, glucosidase I activity of the pooled enzyme fractions from FPLC in the presence of different substrate analogues (1 and 10 mM) was monitored and compared to the activity when such analogue compounds were not added by calculating the percentage enzyme activity remaining. For substrate analogues that were found to have relatively potent inhibitory effect on glucosidase I, a full kinetic evaluation of the mechanism of inhibition was determined. The initial rate data obtained at three different concentrations of the compound over a range of substrate concentrations (0.75, 1.0, 2.0, 4.0, 7.5, 10.0, 12.5 and 15.0 mM) and fit to the Michaelis-Menten equation, using nonlinear regression analysis employing the Marquardt-Levenberg algorithm (SigmaPlot, Jandel Scientific, 1991). 3.3.3. Site-Specific Chemical Modification of Glucosidase I 3.3.3.1. Inhibitory Effect of ED AC on Glucosidase I To determine if there is an acidic amino acid (carboxyl group containing amino acid) 45 present at the active site of glucosidase I, the effect of the carboxyl group specific reagent, ethyl-3-[3-(dimethylamino)propyl]carbodiirnide - EDAC, on glucosidase I activity was examined. E D A C was made up into different concentrations (5, 10, 20, 30, 40, and 50 mM) with pH at 6.8. At each concentration, E D A C was tested to determine whether there was inhibitory effect on glucosidase I activity with various incubation times (30, 45, and 60 min), as described below. A 4 | i l aliquot of E D A C (0, 5, 10, 20, 30, 40, 50 mM) was added into a 500-ul microfuge tube and lyophilized. The microfuge tube was re-constituted with 4 ul of enzyme solution and incubated at 4°C for certain amount of time (30, 45, 60 min). Enzyme activity was then measured using the activity assay and compared to activity of the control (EDAC at 0 mM). A flow chart of the detailed procedure is shown in Fig. 13. The experiments were done in duplicate. In addition, the background absorbance at 450 nm by E D A C , buffer A and H 2 0 was negligible, and E D A C was found not to inhibit the coupling enzymes in the glucosidase I activity assay. 3.3.3.2. Test for Active Site Components of Glucosidase I 10 mM of D N J M was used to protect the active site of glucosidase I (2 ul of D N J M at 40 mM in a 500-ul microfuge tube was lyophilized and re-constituted with 8 ul of the enzyme solution), followed with the addition of 50 mM E D A C (transferred the 8 ul of mixture of D N J M and enzyme solution into a 500-ul microfuge tube in which 2 ul of E D A C at 200 mM had been lyophilized). After removing the D N J M and excessive E D A C by dialysis, activity of the modified enzyme was determined and compared to the activity of enzyme treated with 50 mM E D A C only. Controls without being treated with D N J M and/or E D A C also underwent the same experimental procedure, i.e. with equal time period for 46 CD X> 3 +n CO bfj £ o IH o o o T3 CD N X! a, o o o O cn O CN U < P w <+H o o o CO CD s <D <+H O "3. + c S o cT cn c2 o o T3 CO ts X> o CD T 3 "S X5 O o CO o 3. + l-H <a u o r-cn tS TD <D t« X> 3 CO c SO O -CN o I T ) CN + i-3 <+H O o CN + CD & CO CO CvS o o '§ c3 O T d CD tl CO c c3 a CO 3» CO o o T 3 CD a e o o o o CN •| o cn ccj T3 u o cn ts T3 CD ts JO 3 o d e G O ts T 3 cS CO i-i CO CO O G ccj •e o co Q W o CO "3 O O T 3 CD WH ccj I O o c3 T3 'co O o J 3 "ab G O -4-» o CO o X ! U < 0 w C+H o CO l-H 3 -o CD O 8 PH cn co i-I 47 procedure is outlined in Fig. 14. Briefly, three sets of glucosidase I samples were prepared. The first set of samples were treated with D N J M and later with E D AC. The second set of samples were treated with ED AC only. The third set of samples were used as controls without being treated with D N J M or ED AC. Glucosidase I activity assay was carried out for all three sets of samples at each step during the test. The enzyme activity was recorded and compared. The experiments were done in duplicate and D N J M was found not to affect the glucosidase I activity assay. 48 CJ S Q "5 W -O U 1-1 5 c ^ O T 3 O CU CU u CJ 2 q >, ^H O (U cu to "73 o o O yme solution c <u O H o o -o CU r> assay s) u- <u J3 •s & •a 8 tor CJ 0 CU s w 1^- >s c3 c W <D T3 (U > . 03 CO CO > , <u .-a "H, I a s w >^  a o o 13 60 o e <u co 'cO O o j2 C+H o c o o o £ o e cu O o s cu O. co I 0) c2 u O s OH 49 4. Results and Discussion 4.1. Isolation of Glucosidase I Dry yeast cells were chosen as the source for soluble glucosidase I since they are readily available and have been successfully used elsewhere (Neverova et al, 1994). The isolation of the soluble enzyme was followed with a synthetic trisaccharide a-D-Glc(l->2) a-D-Glc(l—>3)a-D-Glc-0(CH2)gCOOCH3 as substrate. The terminal glucose is released from the substrate by the enzyme and quantitated using glucose oxidase, peroxidase, and o-dianisidine. The enzyme was isolated by a three-step procedure involving ammonium sulfate extraction, Toyopearl D E A E chromatography, and FPLC with a Source Q column. The results of a typical isolation experiment are summarized in Table 4. Crude glucosidase I protein was precipitated with a 20 - 60% ammonium sulfate fraction of the yeast cell supernatant after the ultracentrifugation step and dialyzed against 2 L of buffer A overnight. This fraction of crude protein had a much higher protein concentration and lower glucosidase I specific activity compared to enzyme fractions obtained from later isolation steps, indicating high content of contaminating proteins. This can also be partly shown by the high glucosidase II activity of this crude protein fraction. The high content of impurities resulted in a high background microplate reading of the sample blank of 0.142 A U , compared to a reading of 0.065 A U for the sample blank after FPLC, when assaying the glucosidase I activity, which might have underestimated the enzyme activity of this crude protein fraction since the enzyme activity was calculated based on relatively small absorbance differences in microplate readings between control and sample. This may explain why the total activity of this crude protein fraction is less than that of other enzyme fractions in Table 4. Another possibility is that some of the H 2 0 2 being 50 C+_ r - v G .+4 ofj| CD +3 Xs a . o o >, ° J3 . t i *^ * l r w CD c o .s '3 iT CD »3 S O o 6 CD s ^ o w > a o o ccj ir> CO CN o CN CN CN CO i/o CN CN CO CN 3 co £ 3 o ^ CD H N x t 1 *c3 If _ Q CD CD PL, <D X> IT) CN IT) IT) CO CN CN o IT) co in o 00 in © oo IT) CN 00 CO CO CN CO CO 00 VO IT) a X > h-l g -M PL, N co [T. X ^ 3 *c3 ^ 51 produced was degraded by some other oxidoreductases so that glucosidase I activity was underestimated by the enzyme assay in the crude mixture. Chromatography of the crude protein fraction on a Toyopearl D E A E column increased the purity of the enzyme protein by approximately 10-fold. This step was very critical since it separated glucosidase I from the majority of glucosidase II and other non-specific a-glucosidase activities. The glucosidase II activity (or PNPG activity) dropped approximately 100-fold from the previous step. After three-hour dialysis against 4 L of buffer A, the glucosidase I activity decreased by about 15% which shows that enzyme glucosidase I is very unstable. After this enzyme fraction was subjected to FPLC with a Source Q column, the specific activity of glucosidase I increased approximately by 3 - 4 times from the fraction obtained from the Toyopearl D E A E column. Therefore, this is also an important step since it further decreased the glucosidase II activity by several-fold while increasing the glucosidase I specific activity by several-fold from the previous step. The ratio of glucosidase I / glucosidase II activity after FPLC was around 12/1. Throughout this research, all glucosidase I used for other studies, with the exception of stabilization study where the enzyme was isolated with Combinations A - G (to be discussed in the next section), was isolated using this procedure (Combination D as in the stabilization study). Variation of enzyme activity at each isolation step during each preparation ranged from approximately 3 to 8%. Autolyzing the yeast cells by incubating the yeast cell solution at 37°C for 3 hours instead of passing the yeast cell solution through a French press twice was also tried. However, enzyme isolated with this method had lower activity by 20% to 32% compared to enzyme isolated with a French press. 52 4.2. Stabilization of Glucosidase I Since glucosidase I activity is measured through a series of chemical reactions (Fig. 5), it is critical to ensure that the coupling enzymes and components are not affected by any additives or conditions used. Otherwise, the glucosidase I activity measured will not be very accurate. The effect of the various combinations of additives used to stabilize glucosidase I activity on the enzyme coupled assay was therefore investigated. The results indicated that DTT interfered with the assay (Table 5). This is because the H 2 0 2 produced was reduced by DTT masking the glucosidase I activity. DTT was therefore, not used as an additive in further work. Total activity and percentage yield of glucosidase I isolated with various combinations of additives that could protect the enzyme from inactivation are summarized in Table 6. The results indicated that the total activity of glucosidase I was greatly improved by the use of different additives. The data suggested that the protease inhibitor cocktail, glycerol, PMSF, E D T A and B S A could all help to increase the total yield of glucosidase I. Detailed results including volume, protein content, and enzyme activity of each fraction during isolation steps are in Tables (A - G) in Appendix 1. The effect of different combinations of additives on the stability of glucosidase I was tested by storing the enzyme at 4°C over a period of 30 days, a reasonable time period to determine the trend of enzyme activity loss, and examining the enzyme activity at various times during storage. Among the different trials that were done, glucosidase I isolated with a combination of 10 mM phosphate buffer, 10% glycerol, 0.2 mg/ml EDTA, 100 jam PMSF, and 1 mg/ml B S A (Combination F) had the best stability, while glucosidase I isolated with 10 mM phosphate buffer only (Combination A) was the least stable (Fig. 15). 53 DNJM 0.99 EDAC 0.95 In 0.09 o 0.98 0.95 W 0.94 Q 0.98 U 0.97 ffl 0.96 < 1.00 Solutions « JO rt B CN 54 2 o E E^ o H fl o • M •»-< 05 S 15 S o U PQ 55 (o/0) paurep-a XjiAipy 56 Moreover, the enzyme seemed to lose its activity very quickly during the first week of storage and to stabilize after this. For glucosidase I isolated with 10 mM phosphate buffer only, the enzyme lost most of its activity (around 15% left) in the first nine days of storage. However, since the data was only based on single isolation due to the limited amount of trisaccharide substrate, it was unable to conclude which combination was the best. Based on the data shown, the additives used during the isolation steps, the protease inhibitor cocktail, glycerol, PMSF, E D T A and BSA, could prolong the stability of the enzyme during storage. In practical terms, enzyme used for later studies in this research was isolated with Combination D (10 mM phosphate buffer, 10% glycerol, and 100 u M PMSF) due to the relatively good enzyme activity and stability obtained, its simplicity and low cost. The possible effect of bacteria on the stability of glucosidase I during storage was tested by comparing the stability of sterile and non-sterile enzyme stored at 4°C. The result in Fig. 16 showed that same trend in loss of enzyme activity occurred to both sterile and non-sterile samples throughout the one-month storage. Therefore, it was reasonable to conclude that proteolysis by bacterial contamination did not contribute to the decreased enzyme activity observed in this work under the experimental conditions used. As for freeze drying and freezing treatments, it was shown in Fig. 17 that the stability of enzymes that had undergone freeze drying and freezing treatments behaved very similarly to that of enzyme without any treatment (i.e. stored at 4°C after isolation). The enzyme activity was very unstable for the first week and then dropped down more slowly. Among all samples, the activity of enzymes being frozen at -25°C and -80°C decreased much more gradually over time than that of enzyme that was freeze dried and that of enzyme without treatment. However, freezing temperature did not seem to be an important factor for 57 58 o o o o o o o CN O 00 V£> <N (o/0) paureja-jj AjiAipy 59 enzyme stability, as the enzyme kept at -80°C lost activity at a very similar rate to enzyme kept at -25°C. In addition, about 35% of glucosidase I activity was lost after being freeze dried. The above results indicated that freezing could greatly increase glucosidase I stability during storage (had around 60% activity after a month, compared to having about 40% activity after 30 days when kept at 4°C, after isolation with the same additives -Combination D, refer to Fig. 15) while freeze drying offered little help in terms of retention of activity. 60 4.3. Further Purification of Glucosidase I with Affinity Chromatography 4.3.1. Affinity Chromatography with N-methyl-N-(5-carboxypentyl)-l-deoxynojirimycin as the Ligand Earlier studies demonstrated that 1-deoxynojirimycin (DNJM) is a good inhibitor for a variety of a- and 6-glucosidases including glucosidase I and II (Fuhrmann etal, 1985; Elbein, 1987). Recently it was noticed that the inhibitory effect of D N J M changed dramatically and in the opposite direction for the two enzymes when the ring nitrogen was alkylated. Thus glucosidase I was significantly more inhibited by the N-methyl derivative of D N J M than by D N J M itself, whereas the inhibition of glucosidase II was impaired several-fold by this modification (Bause et al, 1989). Such different effects on glucosidase I and II formed the rationale for the use of N-methyl-N-(5-carboxypentyl)- 1-deoxynojirimycin as the affinity ligand to further purify glucosidase I. In this experiment, glucosidase I activity was found to be absent in the supernatant after being tumbled overnight with the packed gel. It indicated that glucosidase I was either successfully bound to the affinity matrix or had been inactivated during the tumbling. In addition, the purity of the isolated glucosidase I based on specific activity was improved by more than 468 times in terms of specific activity. However, the recovery of total enzyme activity was very low, with only about 0.41%. SDS-PAGE gel also showed that the affinity matrix was not very selective since several bands were observed on the gel. The failure of the experiment might be due to two reasons: 1) tumbling overnight might have denatured the enzyme; and 2) N-methyl-N-(5-carboxypentyl)-1-deoxynojirimycin, the affinity ligand, was not successfully made. 61 4.3.2. Concanavalin A-Sepharose Chromatography In contrast to the above inhibitor affinity chromatography, the use of Concanavalin A-Sepharose chromatography as an affinity resin for purification of glucosidase I was more promising. Although the purity of the isolated glucosidase I was improved by only 6.7 times based on specific activity, the recovery of total enzyme activity reached 32.7%. In addition, only two bands were observed on SDS-PAGE gel (with M W of 92.6 and 79.7 kD respectively) and one of the two bands calculated at 92.6 kD was quite close to the suggested molecular weight of 95 kD for the membrane bound form of glucosidase I (Fig. 18). The molecular mass of soluble form of glucosidase I has not been determined so far. As well, it is not sure as yet if the soluble form of the enzyme is a result of a proteolytic cleavage of the membrane bound form or less likely - the soluble form is an independent gene product, which potentially might not be related to the membrane bound form. Confirmation of the amino sequence of the 92.6 K D band purified from Con A -Sepharose chromatography was also attempted. The detailed procedure, including two steps - electrophoresis and electrotransfer, was as described in Section 3. Every effort was made to avoid interference with the sequencing reaction and to ensure efficient protein transfer. During the electrophoresis step, gel solutions except running buffer, were filtered, with a 0.45 micron filter; samples with solubilizing buffer were heated at 37°C for 10 to 15 minutes prior to loading onto the gel instead of being heated at 100°C for 1 minute; the cast gel was allowed to stand for 24 hours at room temperature prior to use to allow the gel, including stacker, to polymerize completely; 11.4 mg/1 (0.1 mM) thioglycolate was added to the upper running buffer prior to electrophoresis to scavenge reactive compounds left in the gel which could cause N-terminal blocking; and acetic acid was avoided in staining (0.1% 62 £ 2 200 116 97.4 66 45 31 21.5 14.5 6.5 Lane 1 Lane 2 Figure 18. SDS-PAGE pattern of enzyme fraction from Con A-sepharose chromatography. Bands in Lane 1 represent the broad range SDS-PAGE standard, of which molecular weight of each band is showed on the left. Bands in Lane 2 represent the purified enzyme fraction. 63 Coomassie Blue in 50% methanol) and destaining (50% methanol) as it might cause blockage of the amino terminus. During the electrotransfer step, PVDF membrane was completely wetted with 100% methanol and equilibrated with CAPS transfer buffer [lOmM 3-(cyclohexylamino)-l-propanesulfonic acid, 10% methanol, pH 11.0] to ensure proper binding; the high pH of the CAPS buffer assured transfer of most proteins; and CAPS buffer contained no Tris or glycine, which would interfere with the sequencing reaction. However, no clear result was obtained because of many interfering signals present during the amino sequencing analysis. These interfering signals were most probably from glycine used at the electrophoresis step. They could also come from other protein impurities mixed with the purified glucosidase I. In addition, N-terminal blockage might have still occurred during the sequencing. Reducing agents such as 2-mercaptoethanol or dithioerythritol were not added to buffers used for the isolation and purification steps since they could interfere with the glucosidase I activity assay. Lack of such reducing agents could lead to formation of aggregates through disulfide bridge formation and the attack of the amino group by radicals, aldehydes or oxygen, which in turn may have led to N-terminal blockage. 64 4.4. Stereochemistry of Glucosidase I Reaction The stereochemical course of glucosidase I reaction can be determined using 1 H N M R spectroscopy to directly monitor the release of glucose from the synthetic trisaccharide substrate aGlc(l-2) aGlc(l-3)aGlc-0-(CH 2)8COOCH 3 . The time course of hydrolysis catalyzed by the yeast glucosidase I is shown in Fig. 19 where partial spectra of the anomeric region are shown. The signals corresponding to the anomeric protons of each of the glucose residues in the trisaccharide substrate, H I " at 5.54 ppm, H I ' at 5.17 ppm and HI at 4.91 ppm and disaccharide product H I ' at 5.34 ppm were well resolved from each other and from free glucose. Three minutes after the addition of enzyme, the anomeric proton of P-ghicose was present at 5 = 4.64 ppm, as well as disaccharide product H I ' signal at 5.34 ppm. The J3-glucose - HI signal reached the 5% level after 13 minutes. This level was reached by the a-anomer - HI after 53 minutes, indicating a lag time of 40 minutes. The yeast a-glucosidase I reaction therefore occurs with inversion of anomeric configuration. After 5.5 hr the H I " trisaccharide as well as H I ' trisaccharide signal disappeared into the noise of the spectrum while the buildup of H I ' disaccharide was completed. In addition, the disaccharide product (after one glucose was cleaved from the trisaccharide) was built up but no conversion to monosaccharide was seen. It indicates that glucosidase II does not appear to be present in the isolated glucosidase I, or the amount of residual glucosidase II does not seem to be enough to hydrolyze a significant amount of disaccharide in the time period of the experiment or the disaccharide is a poor substrate. Since the above results prove that glucosidase I is an inverting enzyme, and the carboxylic side chains of aspartic or glutamic acids have been implicated in the catalytic mechanism of other inverting glycosidases, it is likely that at least one acidic amino acid 65 J L J l J l 1 1 1 1 1 1 1 H1'(dis.) 1 A A J V -A\_ J*_A i H1 I aGlc-H1 f M H1" | MH1-(tri,) M M Jl JL Jl 1 1 1 A PGic-m L 5:30 4:30 3:30 Jf< IL 2:30 ^ 2:00 / V I I J L i : 3 0 1:00 0:33 0:28 0:23 0:18 0:13 0:08 0:03 -i 1—i—i—i—r-|—i i i—r-|—i—i—i—i—|—t—i—n—| I i—i—p—|—rn—i—r—[—r-r 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 I 1 1 1 ' T ppm ure 19. Time course of glucosidase I reaction (Palcic et al., 1999). 66 which contains a carboxyl group is specifically involved in the active site of glucosidase I. 67 4.5. Kinetic Evaluation of Glucosidase I Substrate Analogues Inhibitory effects of 11 synthetic substrate analogues were investigated by calculating the percentage enzyme activity remaining when these substrate analogues were added into the isolated enzyme. For substrate analogues that were found to have relatively potent inhibitory effect on the enzyme, the K m (apparent) and V m a x values were obtained using a direct fit of velocity and substrate data to the Michaelis-Menton equation, using nonlinear regression analysis. By re-plotting K m (apparent) against substrate analogue concentration, K; value of the substrate analogue,was calculated as the negative x intercept of the re-plot, obtained by linear regression. Among the glucosidase I substrate analogues tested in this research, 2'Fluoro-aGlc(l-2)aGlc-0-(CH 2)8COOCH 3 and 2 'N 3 -aGlc( l -2)aGlc-0-(CH 2)8COOCH 3 showed the most potent inhibitory action against glucosidase I (decreased -60% at 10 mM), while the others were less effective inhibitors (Table 7). There was no effect on the glucosidase I coupling assay components, when the analogues were present at 1 and 10 mM. Results of the kinetic evaluation of these two compounds are shown in Figs.20 and 21 respectively. It was indicated that V m a x values calculated from reactions with different substrate analogue concentrations were approximately equal while K m values increased with increasing substrate analogue concentrations. Therefore, the mechanism of inhibition is suggested to be competitive type, i.e. 2'Fluoro-aGlc(l-2)aGlc-0 - (CH 2 ) 8 COOCH 3 and 2'N 3 -aGlc(l-2)aGlc-0-(CH 2 ) 8 COOCH 3 are both competitive inhibitors of glucosidase I. In addition, a linear relationship between the apparent K m and inhibitor concentration exists for these two compounds (Figs. 22 and 23), from which the K; values of 2'Fluoro-aGlc(l-2)aGlc-0-(CH 2) 8COOCH 3 and 2'N 3-aGlc(l-2)aGlc-0-(CH 2 ) 8 COOCH 3 were calculated to be 0.67 mM and 0.80 mM respectively. Due to their 68 00 S3 •g s CD IH > > H-* ;> *+-* o cd r—I <D CO Cd IH 'co O CJ 3 5 CN »—H CO CO CN r - 00 rt TT CN ON CN t-- m in m o '—1 VO t—i CN i—i CN r-i r-I r-i CN CN rt -H -H -H -H -H -H -H +\ -H -tl -H m 00 00 CO TT in TT VO CO VO rt m CN IT) 00 CO TI- o Tt- VO m Tr-vd vd O N CO iri ON r-' CN r-i o od 00 O N VO r-- vO 00 m C-- m CO VO CN t-- r- rt rt VO CN vo r-00 CN O N r-l o ON VO CN 00 CO r-CN r-l r^ CN CN r-i r-i r-i o rt r-i -H -tl -H -H -H -H -H -H -H -H -H m CO o r-~ TT r- vo CO 00 vo 00 O 00 VO CN o m 00 VO CO r - m CN r--' t--' vd ON in ON Tf' O N rt m ON ON ON c-- O N 00 r- ON O N CO CD 3> o c cd u 13 i_ +-> CO 3 C/N cc! <D u-00 i o I o cd CD l_ 00 6 i o CD co cd <D CD CO cd CD oo &o O I o I o 6 C; s 5 ^ ^ ^ ^ CN CN CN" CN cd CD I-. 60 • O CD CO cd CD t-60 i o 8 8 o CD Q CO o CD Q cj O o 5 8 8 iV £ o "S CD >*H p ^ vo O O CJ 5 5 CN" CN" I I T—H rt 5 o 8 8 t3 i? 6 CN o Tf CD CO cd <D i_ 60 • o I o i 5 8 CN CD CO cd CD l_ 60 • o cj CD CO cd CD u. 60 6 i cj o o 8^ <3 CN" CN" a o 8 8 o o o l-H o . . 2 CJ u vb vo CD co cd CD LH 60 • o 6 5 & CN i "cj 5 8 • o IH O 3 E CN 69 (pura / jq) /CjioopA/T 70 (pura/jq) XjioopA/T 71 (J\[UI) (;u9JBddr>) "Sj 72 (p\[ui) (;u9jr>ddr>) "Hj 73 relatively less potent inhibitory effect on glucosidase I (50% inhibition achieved at about 10 mM), compared to deoxynojirimycin which inhibits the enzyme activity by 50% at 50 u M (at substrate concentration of 0.4 K m ) (Neverova et al, 1994), the two compounds in the present study would not be useful as therapeutic agents. 74 4.6. Site-Specific Chemical Modification of Glucosidase I The effects of various concentrations of ED AC on glucosidase I activity are shown in Fig.24. Increasing glucosidase I inhibition was found as the concentration of E D AC increased from 10 to 50 mM, and inhibition also increased with incubation time, indicating that the primary structure of glucosidase I is composed of carboxyl group containing amino acids. In order to investigate further whether carboxyl group containing amino acids are present at the active site of the enzyme, ED A C at 50 mM which showed the most inhibitory action (about 90% inhibition when incubated for 60 min) in Fig.24 was selected for the next experimental procedure. In the next experimental procedure, glucosidase I was treated with 10 mM D N J M and/or 50 mM ED AC. The results are shown in Figs.25 and 26. Glucosidase I activity decreased to about 8.5% of the original after addition of 10 mM DNJM. A slight decrease (less than 3%) in enzyme activity was observed when 50 mM ED A C was added into the enzyme and D N J M mixture. However, about 60% of the original enzyme activity were recovered after dialysis overnight during which D N J M and the excess ED A C were removed. When 50 mM ED A C was added to this recovered enzyme activity, it was again inhibited to about 10%. Therefore, it seemed that D N J M could protect the active site of the enzyme so that ED A C could not freely interact with the amino acid residues at the active site. The interaction between D N J M and the active site residues was reversible. On the other hand, dialysis overnight did not help to recover enzyme activity when the enzyme was treated with 50 mM ED A C only. Such modification was irreversible under the conditions used in this experiment. ED A C is one of the water-soluble carbodiimides that have been widely used to 75 a a • rH S3 o B T> © II Tt-II vo II II s II g II a ft H H H a e a o o o \£-i •.jri es re td X> X x> 3 3 o o o C Ci C i — i r—1 HI o C N O T i Tt-o o C O T l C N © C N C U 60 cd i_> CD £ CD xs •*-» 60 CD P S3 ' a * •a ^ cd cu P o o 1/1 1— 3 X> p CD co CD kH 4-* s ^ 00 CD .5 3 ^ S T3 t H CD ^ O td S b "3 PM cd S ? 1 | 1 » § S ~" CD 60 _ P O cd l-H V-I r> U > '> x> £ 8 g s O CD i?T> P <D g cd 6£CD co rt SR C "53 «S £ O 3 3 o x> 3 CD ,_ 60 cd O p -S tl 0 C P U cS «H ° 2 CO 2 Q o S i I 1 CD td co CO CO rt CD td O QH (JOJJU03 jo %) AjiAipy auiAzug Suiureuia^ 76 AjiAuoy renpisffjj % 77 a c3 •3 tz> 78 modify carboxyl groups in proteins. The reaction of carbodiimides with protein carboxyl groups can be seen in Fig.27 (Lundblad, 1995). The reaction sequence is initiated by addition of the carboxyl across one of the double bonds of the diimide system to give an O-acylisourea. The activated carboxyl group of this adduct can then react via one or two routes. First, an attack by a nucleophile will yield an acyl-nucleophile product plus the urea derived from the carbodiimide. Second, the O-acylisourea can rearrange to an N-acylisourea via an intramolecular acyl transfer (Khorana, 1953). Although modification of the carboxyl group in proteins with a water-soluble carbodiimide has proven to be extremely useful, there are problems with side reactions (Carraway and Koshland, 1972). Carbodiimides are not specific for carboxyl groups alone. Under the conditions employed in this investigation, ED A C would also react with aromatic hydroxyl groups of tyrosines and sulfhydryl groups of cysteines. Since the rate of reaction between tyrosine and ED A C is much slower than that between carboxyl groups and ED A C (Carraway and Koshland, 1968), and D N J M has been showed to be unable to protect glucosidase I from inactivation by tetranitromethane, a reagent specific for tyrosine (Romaniouk and Vijay, 1997), tyrosine is not likely to be present at the active site of glucosidase I. As for cysteines, Pukazhenthi et al. (1993) have suggested that a reactive cysteine was at or near the active site of glucosidase I. However, this does not deny the existence of a carboxyl group at the active site. It is also possible that at least a carboxyl group is involved in the inhibitory effect of E D A C on glucosidase I. There are examples of both cysteine and carboxyl groups being present at the active site of different glycosidases and the carboxyl amino acids playing the catalytic role have been found. Takayanagi et al. (1995) showed that both carboxyl and sulphydryl groups are present at the active site of beta-fructofuranosidase (E.C. 3.2.1.26) and both are essential 79 Protein-COOH + R-N=C=N-R' O H N - R ' II II *• Protein-C-O-C NH-R / O-acylisourea O II + H 2 N - C H 2 C - O C H 3 0 0 O II II II P r o t e i n - C - N H - C H 2 C - O C H 3 + R - N H - C - N H - R ' O II O C - N H - R ' II II protein-C-N R N-acylisourea Figure 27. The reaction of carbodiimide with protein carboxyl groups. 80 for enzyme activity. Therefore, the result of modification of glucosidase I active site by ED A C in this research work either supports the presence of cysteine at the active site, or provides evidence that at least one acidic amino acid is located at the active site. 81 5. Conclusions Glucosidase I is very unstable during isolation and storage under normal conditions (e.g. at 4°C). In this research, the stability of the enzyme was much improved by the addition of glycerol, EDTA, BSA, PMSF and protease inhibitors during isolation. When kept at extremely low temperature (-25°C and -80°C), the enzyme lost activity less rapidly than when stored at higher temperature (4°C). Freeze-drying treatment did not help to retain enzyme activity during storage. Glucosidase I is shown to be an inverting enzyme which catalyzes hydrolysis with inversion of anomeric configuration and it can be further purified with Con-A chromatography. Kinetic study shows that substrate analogues 2'Fluoro-aGlc(l-2)aGlc-0-grease and 2'N 3-aGlc(l-2)aGlc-0-grease are competitive inhibitors of the enzyme. However, these two compounds would not be useful as glucosidase I inhibitors due to their relatively less potent inhibitory effect (only 50% inhibition at about 10 mM). In addition, modification of glucosidase I active site by ED A C either supports the presence of cysteine at the active site or provides evidence that at least an acidic amino acid is specifically involved in the active site of glucosidase I. Although studies focused on the identification of the protein structure, the catalytically important active site residues, cloning and expression of the enzyme, and the role of the enzyme in the biosynthesis of cell wall components have begun, more work is needed in order to better understand the catalytic mechanism of glucosidase I and develop inhibitors as antiviral (HIV) and antitumor therapeutic agents. 82 6. References Abeijon, C. & Chen, L . Y . (1998). The role of glucosidase I (Cwh41p) in the biosynthesis of cell wall B-l,6-glucan is indirect. Moi. Biol. Cell 9: 2729 - 2738. Aleshin, A.E. , Firsov, L . M . & Honzatko, R.B. (1994). 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In: Techniques in Protein Modification, pp.233, CRC Press, Boca Raton, FL. Mikami, B. , Hehre, E.J., Sato, M . , Katsube, Y. , Hirose, M . , Morita, Y . & Sacchettini, J.C. (1993). The 2.0A resolution structure of soybean beta-amylase complexed with alpha-cyclodextrin. Biochemistry 32: 6836 - 6845. Montefiori, D . C , Robinson, W.E., Jr. & Mitchell, W.M. (1988). Role of protein N -glycosylation in pathogenesis of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 85: 9248 - 9252. Neverova, I., Seaman, C.H., Srivastava, O.P., Szweda, R., Vijay, L K . & Palcic M . M . 87 (1994). A spectrophotometric assay for glucosidase I. Anal. Biochem. 222: 190 - 195. Niwa, T., Inouye, S., Tsuruoka, T., Koaze, Y . & Niida, T. (1970). Nojirimycin as a potent inhibitor of glucosidase. Agric. Biol. Chem. 34: 966 - 968. Palcic, M M , Seaman, C.H., Otter, A., Szpacenko, A., Romaniuk, A., L i , Y . X . & Vijay, L K . (1999). Processing a-glucosidase I is an inverting glycosidase. 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Approaches to labeling and identification of active site residues in glycosidases. Prot. Sci. 4: 361 - 372. 91 A p p e n d i x 1: Results of isolating glucosidase I with various combinations of additives including volume, protein content and enzyme activity of each fraction during isolation steps. 92 o •£ a \% o ca O © >/-> NO 00 o CN CN ON 9 HH 5 s H "8 .9 <=> N O " © NO CN 00 ON NO o 00 Sa ft fo i> ON - * NO as TT ND O © 00 . 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