Mechanistic studies on the cellulomonas fimi exoglycanase (Cex)
Creators: name: Birsan, Camelia
Publisher: name: University of British Columbia
Year: 2009

description: The objective of this study was to probe the roles of the conserved residues His205 and Gln87 in the active site of the retaining exoglycanase (Cex) from Cellulomonas fimi, a member of the family 10 glycosyl hydrolases. Cex has been previously shown to hydrolyze ß-1,4 glycosidic bonds in xylan, cellulose, and a range of soluble aryl-glycosides by a double displacement mechanism via formation (glycosylation) and hydrolysis (deglycosylation) of a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis of the enzyme reveals that His205 is positioned within hydrogen bonding distance of the catalytic nucleophile, Glu233, suggesting that it may play a role in modulating the ionization state of this key residue. Structural analysis also reveals that Gln87 is located within close proximity of the C-5 hydroxymethylene group of the distal glucose unit of a cellobioside substrate bound in the active site of Cex, thus suggesting a potential role of Gln87 in substrate binding and specificity. His205 has been systematically replaced with alanine and asparagine via sitedirected mutagenesis, and the resulting effect on overall structure, activity and pH profile has been determined. Kinetic analysis of mutants revealed that His205 is required for both the glycosylation and the deglycosylation steps; rate reductions of 300-5600 fold for His205Ala and 2000-9000 fold for His205Asn were observed on the glycosylation step, while the rate reductions on the deglycosylation step were found to be 4000-5000 fold for His205Ala and 8000-9000 fold for the His205Asn mutant. These effects are consistent with the proposed role of His205 in maintaining the proper ionization state of Glu233. Additionally, His205 may provide some acid catalytic assistance to the oxygen of the leaving group aglycone at the glycosylation transition state, thereby explaining the larger Br0nsted constants measured on the glycosylation step for the His205 mutants than the wild type enzyme. During the deglycosylation step, His205 may be important in stabilizing the developing negative charge on the carboxylate group of the departing enzymic nucleophile and enhancing the nucleophilicity of the attacking water molecule, as suggested by the smaller secondary deuterium kinetic isotope effect on the deglycosylation step for the His205Asn mutant. The pH dependence of the glycosylation step showed that ionization states of key catalytic residues in the free enzyme were perturbed upon mutating His205 to either alanine or asparagine, indicating that His205 is important in maintaining the proper electronic environment in the active site of Cex. In order to compress the binding pocket of the C-5 hydroxymethylene groups of substrate to reduce the cellulase activity of Cex, Gln87 has been substituted with the bulkier residues methionine, tyrosine and histidine via site-directed mutagenesis. Kinetic investigation of the Gln87Met mutant revealed no significant changes in the substrate specificity relative to that of the native enzyme, and therefore the function of Gln87 in substrate recognition is not clear. It is hoped that future research activities will provide more insights into this question.