Insights into the catalytic mechanism of a retaining xylanase from Cellulomonas fimi

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 beta-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 beta-xylobioside were determined, and very good correlations were observed in logarithmic plots relating the Ki value for the sp2-hybridized class of inhibitor with 10 mutants and kcat/Km for the hydrolysis of the substrate by the corresponding mutants. The dependence was significantly less in the plot of log(kcat/ Km) versus log(1/Ki) for the sp3-hybridized class of inhibitor, indicating that the sp 2-hybridized class of inhibitors more closely mimics the geometry of the transition state than does the sp3-hybridized class of inhibitors.