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Manipulating the locations or presences of carboxylic acids in glycosides can significantly change their mechanisms. Mutagenesis may, when coupled with effective screens, provide the most effective approach to altering specificities.

The binding of various active site mutants of Bacillus circulans cyclodextrin glycosyltransferase to the inhibitor acarbose was investigated. The active site mutants were screened using two substrates, namely, alpha-maltotriosyl fluoride and alpha-glucosyl fluoride. Experimental results showed that significant correlations exist in logarithmic plots relating the Ki value for acarbose with each mutant and both k(subscript cat)/K(subscript m) and K(subscript m) for the hydrolysis of either substrate by the corresponding active site mutants.

Glycosidases are of interest due to their role in many biological and industrial processes. They are classed as 'retaining' or inverting' and both types have two catalytic carboxylic acid residues. Structural information is a key requirement to understanding the enzymes and affinity labels have been successfully used to identify the acid/base catalysts in certain cellulases and xlylanases. Novel cellobioside and glucoside affinity labels have been synthesized, using chemoenzymatic methods designed for labelling enzyme acid/base catalysts, incorporating an alpha-bromoketone functionality.

An analysis of the mechanism of the Cel5A, a retaining cellulase from Bacillus agaradhaerens, was conducted. X-ray crystallography was used in the structural analysis of Cel5A in all its stable states along the reaction coordinate. Results from the kinetic analysis of the formation and hydrolysis of the intermediate as well as the structural results demonstrate the catalytic mechanism of retaining glycoside hydrolases. Data also provide an insight into the utilization of binding energy in enzymatic catalysis.

Time-resolved electrospray ionization mass spectrometry (ESI-MS) has been applied to the measurement of the kinetics of the reaction catalyzed by the xylanase of Bacillus circulans by focusing on the kinetics of formation of a transient enzyme intermediate. Xylanase catalyzes the beta-xylobiosides via a double-displacement process that passes through a covalent xylobiosyl-enzyme intermediate. Time resolution of the ESI-MS process allows for the measurement of the pre-steady state kinetics of the covalent intermediate formation.

The crystal structure of the substrate-enzyme intermediate, 2-deoxy-2-fluoroxylobiosyl-Cex, was determined and compared with unliganded structure of the enzyme and the covalently bound enzyme-substrate crystal structure to identify the factors that contribute to the higher binding affinity of the beta- glycanase enzyme Cex to xylan. Results reveal that the glutamine residue at position 87 and the tryptophan residue at position 281 are important determinants of enzyme specificity owing to their contribution to the conformation of the catalytic domain.

A study was conducted on the synthesis of alpha-C-mannoside and alpha-C-glucoside, the ability of alpha-C-mannoside to inactivate various alpha-glycosidases. The study also addressed the labeling and identification of the postulated acid/base catalyst of yeast alpha-glucosidase. The results indicate that Glu-276 is situated within the active site of yeast alpha-glucosidase and that this residue plays a key role in catalysis.

The substrate 4-deoxymaltotriosyl alpha-fluoride and the acid/base catalyst mutant Glu257Gln are used to trap and characterize cyclodextrin glycosyltransferase. Edman sequencing method is used to identify reaction intermediates in the said process. Results identified Asp229 as the catalytic nucleophile. Evidence showing the existence of a covalent intermediate in CGTase was also unearthed.

The interactions between beta-glucosidases the sugar glycon was investigated through kinetic analyses of the beta-glucosidase in Agrobacterium faecalis. Results have shown that accumulation of 2-deoxy-2-fluoroglycosyl-enzyme resulted in inactivation of the of the enzyme. Interactions between the enzyme and the sugar aglycon provide sufficient transition state stabilization, allowing formation and trapping of the glycosyl-enzyme.

Research shows that the catalytic nucleophile in the Fusarium oxysporum enzyme is Glu197. The hydrolysis of cellulose is catalyzed by Fusarium oxysporum's endoglucanase EG I through a double-displacement mechanism in which a glycosyl-enzyme intermediate is formed and hydrolyzed. Glu197 was identified in a 2-fluorocellobiosyl-labeled peptide by analyzing the peptic digest of an inactivated enzyme, with Edman degradation analysis and comparative mapping studies confirming the find.

Bacillus circulans xylanase was used as a model system to study the effect of acid/base catalyst and its spatial positioning on the functional activity of glycosidase. The results of kinetic studies involving B. circulans xylanase bearing Glu172Asp mutation confirmed that the hydrolysis of xylan is greatly influenced by the distance between the two catalytic carboxylic acids of the enzyme. The enzymatic activity was completely abrogated with the removal of the carboxyl group although the absence of such acid/base catalyst can be compensated for by the addition of an anionic nucleophile in the reaction system. The results thus confirm the role of Glu172 as an acid/base catalyst and the importance of carboxyl groups at such position.

The role of the essential cofactor pyridoxal phosphate in rabbit muscle glycogen phosphorylase catalysis was examined. Two phosphonate analogues of pyridoxal phosphate were prepared and reconstituted into apophosphorylase b. Findings revealed significant changes in phosphate environment upon nucleotide activation. Both such reconstituted enzymes had activities of about 25-30% of that seen in the native enzyme, while Km values were similar to those of the native enzyme. Results indicated that pyridoxal phosphate does not act as an essential acid/base catalyst in glycogen phosphorylase.

Steady-state kinetic studies of the conserved active site carboxylic acids in the exoglucanase/xylanase Cex from Cellulomonas fimi suggest that these carboxylates function in general acid/base catalysis. Elimination of the catalytic nucleophile diminishes the catalytic properties of the enzymes. In contrast, the addition of small anions restores activity, but as an inverting enzyme.

The position of the two carboxyl groups in Bacillus circulans xylanase has an important effect on its catalytic action. Mutagenesis of the catalytic nucleophile Glu78 helps to change the distance between the two groups. The rate of glycosylation reduces drastically due to an increase in the separation. Catalytic activity reduces to a lesser degree when there is a decrease in the separation. The rate differences are not reflected in the extent of bond cleavage or proton donation at the transition state. Removal of the carboxyl group (Glu78Cys) stops catalytic activity.

Selective isotopic labelling and 13C-NMR studies on the pK(sub a) values of the catalytic glutamic acid residues in wild type/mutant Bacillus circulans (BCX) and subtilis xylanases reveal a two-step hydrolysis mechanism involving a covalent glycosyl-enzyme intermediate. The double-displacement reaction of BCX may be broken into glycosylation and deglycosylation steps. The glycosylation step produces the glycosyl-enzyme intermediate and deglycosylation regenerates the native enzyme. These protonation and deprotonation steps are governed by glutamic ionization states.

The glycogen-debranching enzyme plays a key role in the degradation of glycogen. It works in tandem with glycogen phosphorylase by catalyzing the removal of branching from glycogen. This debranching process, which occurs in two steps, begins with the transfer of a maltotriosyl unit to the main chain and then proceeds with the hydrolysis of the residual glucose moiety. An examination of this process shows that Asp 549 serves as the catalytic nucleophile of the glycogen-debranching enzyme.

Steady-state and pre-steady state kinetic analysis of the hydrolysis of a series of monosubstituted aryl glycosides was conducted to examine the role of noncovalent interactions in the catalytic mechanism of the Agrobacterium faecalis beta-glucosidase. Relatively weak contributions of each hydroxyl group to the binding of these substrates at the ground state were noted, but were observed to be much greater at the two transition rates of glycosylation and deglycosylation. The most significant transition state interactions were at the 2 position, followed by the interactions at the 3 and 6 positions.

The substrate 2,4-dinitrophenyl beta-D-glucopyranoside inhibits the hydrolysis of a covalent alpha-D-glucopyranosyl-enzyme intermediate by the Glu358Asp beta-glucosidase mutant from Agrobacterium. The substrate inhibition is time-dependent. The substrate-induced enzyme inactivation is probably the consequence of occasional derivatization of an alternative nucleophilic group in the active site of the mutant enzyme, since the original Glu358 nucleophile is mutated. The Tyr298 in the enzyme orients Glu358 and stabilizes the deprotonated state of the non-mutated enzyme.

Circular dichroism spectroscopy reveals that structures of the mutants of endoglucanase gene CenA from Cellulomonas fimi, prepared by the replacement of Asp 216, Asp252, Asp287 and Asp 392 by alanine and glutamate, are similar to those of wild-type enzymes. Mutation exerts no influence on the active site structure. Examination of the catalytic ability of these mutants reveals that catalysis does not require Asp 216, and Asp287 cooperates with Asp252 in acid catalysis.

Kinetic studies of the hydrolysis of 2-acetamido-D-glucal by three beta-N-acetylhexosaminidases reveal that the formation of a reversible complex acting as a transition state causes the observed tight binding. The anomeric configuration of the glucal does not change during hydrolysis. Protonation from the top face occurs during the hydration reaction, indicating that glycals with appropriate substituents can serve as intermediates in these reactions by retention of beta-glycosidase.

Determination of the high-resolution crystal structure of the Cex catalytic domain in Cellulomonas fimi reveals that the crystal structure contains 2400 protein atoms, 0.217 R-factor and 45 water molecules at a resolution of 1.8 Angstrom units. The folding of the protein into an eight-parallel-stranded alpha/beta-barrel is new for the microbial beta-glycanase Cex. While the active site exists as an open cleft on the alpha/beta-barrel carboxy-terminal, the hydrogen bonding network in the domain stabilizes the ionization states of vital residues for regulating the equilibrium of the reactions.

Electrospray mass spectrometry determined glutamic acid 78 as the active site nucleophile in the xylanase of Bacillus subtilis. 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside, which inactivates Bacillus subtilis xylanase, is used with the new tandem mass spectrometric method to detect glutamic acid 78. Carboxyl groups of glu78 and another glutamic acid located in the active domain are separated by a distance of 7 angstrom units.

Application of aryl B-D glycoside substrates enabled a kinetic examination of the double displacement mechanism adopted by the enzyme exoglucane/xylanase from cellulomonas fimi (Cex) to hydrolyze its substrates. The xylobiosides and glucosides and Broensted relationships were established for the substrate types. The important properties of the distal sugar were greater nucleophile preassociation, irrespective of the decomposition rate, and an increase in the rate of glycosyl enzyme intermediate formation by improved acid catalysis.

A kinetic study establishes Glu127 as the ideal acid/base catalyst in the hydrolysis of B-1,4-glycosidic bonds by exoglucanase/xylanase Cex from cellulomonas fimi by a double displacement mechanism. This mechanism involves the formation of a glycosyl enzyme intermediate which gels hydrolyzed with the help of a general acid/base catalyst. Mutants were constructed after replacing glutamic acid with alanine. Glu127 exhibited the sequence alignments of the required acid/base catalyst. Azide reacted rapidly with glycosyl-enzyme intermediate, formed the glycosyl azide product, increased the site rate and occupied the vacant anionic site created by acid/base catalyst removal.

Site-directed mutagenesis was used to investigate the role of the active site nucleophile Glu 358 in Agrobacterium beta-glucosidase. Complete inactivation of beta-glucosidase resulted from substitution with Asn and Gln while low activity resulted from replacement with Asp. The mutations significantly hindered binding of transition-state analogues which implies a role for Glu 358 not in substrate binding but in transition-state stabilization. In addition, glycosylation was found to be the rate determining step for all these substrates. Furthermore, Glu 358 stabilizes the cationic transition states and yields covalent intermediates.

The inactivation of Agrobacterium beta-glucosidase by 2-deoxy-2-fluoroglycoside through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-glucopyranosyl enzyme intermediate was thoroughly investigated. Simultaneous release of aglycon during inactivation was observed. In addition, the rate of inactivation depended on the leaving group ability of the aglycon, which yields only partial inactivation due to competition between turnover and formation of intermediates. Moreover, the turnover rate was increased by transglycosylation to a suitable glycoside bound in the aglycon site, yielding a disaccharide product.

Kinetic studies were conducted on beta-glucosidase from Agrobacterium faecalis. It was found to possess a relatively broad specificity for the substrate sugar moiety. In addition, beta-glucosidase exhibited pH-dependent inactivation by cyclophellitol, a mechanism-based inactivator. Moreover, the results provide evidence for a two-step mechanism involving a glucosyl-enzyme intermediate formed and hydrolyzed through oxocarbonium ion-like transition states.

Fluorination of three aldose (D-glucose, alpha-D-mannopyranosyl and 2-deoxy-2-fluoro-alpha-D-glucopyranosyl) phosphates results in the synthesis of their corresponding phosphofluoridates. Along with the synthesized cyclic analogue of 6-phospho-alpha-D-glucopyranosyl phosphate, the phosphofluoridates are tested as inactivators of phosphoglucomutase, an important metabolic enzyme. No such inactivation is observed, but the four phosphocarbons exhibit reversible competitive inhibition of the enzyme. Possible reasons for the failure of inactivation, especially in the light of other documented experimental results, are discussed.

19F nuclear magnetic resonance was used to examine the complexes of phosphoglucomutase with fluorinated substrate analogues. Also, the effects Li+- and Cd2+-binding to these complexes were determined. The binding of inhibitors to the phosphoenzyme induced very large downfield chemical shift changes while smaller shifts were observed for ligands substituted with fluorine at other positions. The results suggest an exchange type of mechanism in which two distinct glucose ring binding sites are postulated.

Kinetic studies were conducted to obtain estimates of hydrogen bonding contributions of each of the sugar hydroxyls to the binding of the substrate alpha-D-glucopyranosyl phosphate both in the ground state and at the transition state for the initial phosphoryl transfer. Slight strengthening of the interactions at each of the hydroxyls were observed only upon progressing from the ground state to the transition state. Moreover, all the glucose-1-P analogues, with the exception of those substituted at position 6 , were found to be substrates for phosphoglucomutase.