The family 10 xylanase from Cellulomonas fimi (Cex) is an important model enzyme on which numerous mechanistic studies have been performed. This enzyme catalyzes the hydrolysis of β-glycosidic linkages via a double-displacement mechanism involving the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate with net retention of stereochemistry at the centre undergoing substitution. The finer details of the mechanism of this enzyme were investigated in three studies in order to gain a better understanding of this family of enzymes. In the first study presented in Chapter 2, the roles of key active-site residues in the catalytic mechanism of Cex were investigated by utilizing site-directed mutagenesis in combination with steady state kinetic analyses and pH-rate dependencies. The rate-determining step for the aryl substrates tested remains deglycosylation for many of the enzymes, while the altered pH profiles demonstrate a role for these highly conserved residues in the hydrogen-bond network responsible for maintaining the ionization state of the two catalytic residues. In Chapter 3, a second study addresses a fundamental enquiry of mechanistic enzymology; that is, how distal and proximal substrate interactions influence catalysis. By systematically removing hydrogen-bonding interactions through modification, individually, of substrate and enzyme, deep insight is gained into the effects of these modifications on each step of the hydrolysis reaction catalyzed by Cex and a family 11 xylanase (Bcx). The data obtained provide significant insight into the contributions of hydrogen-bonding interactions at the distal and proximal sites. The strongest bond energies were measured in the proximal site, suggesting that these interactions are critical for substrate binding and bond hydrolysis. A particularly important finding of this study is that both 'uniform' and 'differential' binding interactions are recruited in the active site of a single enzyme. The third study, presented in Chapter 4, examines how well a series of five high affinity inhibitors mimic the transition state of Cex as a function of the sp²- or sp³ -hybridization state of the "anomeric carbon". Kinetic parameters for o-nitrophenyl β-xylobioside were determined, and very good correlations were observed in logarithmic plots relating the K[sub i] value for the sp² -hybridized class of inhibitor with 10 mutants and k[sub cat]/K[sub m] for the hydrolysis of the substrate by the corresponding mutants. The dependence was significantly less in the plot of log(and k[sub cat]/K[sub m]) versus log(1/K[sub i]) for the sp³-hybridized class of inhibitor, indicating that the sp²-hybridized class of inhibitors more closely mimics the geometry of the transition state than does the sp³-hybridized class of inhibitors.Science, Faculty ofChemistry, Department ofGraduat