The exoglucanase/xylanase Cex from Cellulomonas fimi hydrolyzes beta-1,4-glycosidic bonds with net retention of anomeric configuration, releasing the disaccharides beta-cellobiose or beta-xylobiose. It uses a double-displacement mechanism involving a glycosyl-enzyme intermediate which is formed and hydrolyzed with general acid/base catalytic assistance. Glu127 was proposed as the acid/base catalyst on the basis of sequence alignments, and mutants at this position were constructed in which the glutamic acid is replaced by alanine or glycine. The following kinetic analysis provides firm support for the assignment of Glu127 as the acid/base catalyst and suggests a more general strategy for identification of this residue in other glycosidases. Substrates which do not require protonic assistance for initial bond cleavage exhibit kcat/Km values similar to those of wild-type enzyme, whereas substrates which do require assistance have kcat/Km values over 6000-fold smaller. Thus rate constants for glycosylation are affected to different degrees by this substitution, depending upon their need for acid catalysis. The deglycosylation rate constant is decreased 200-fold by such substitution, due to the removal of general base catalytic assistance. In the presence of sodium azide a new product, beta-cellobiosyl azide, is formed with these mutants whereas only cellobiose is formed with wild-type enzyme or the Glu127Asp mutant under similar conditions. Addition of azide results in very significant increases in kcat values, ranging from 8-fold for 4''-nitrophenyl cellobioside to over 200-fold for 2'',4''-dinitrophenyl cellobioside, whereas kcat/Km values for these substrates remain essentially constant. No effects on rate upon azide addition are seen with substrates containing aglycons of poor leaving group ability.(ABSTRACT TRUNCATED AT 250 WORDS)

The exoglucanase/xylanase Cex from Cellulomonas fimi is a retaining glycosidase which functions via a two-step mechanism involving the formation and hydrolysis of a covalent glycosyl-enzyme intermediate. The roles of three conserved active site carboxylic acids in this enzyme have been probed by detailed kinetic analysis of mutants modified at these three positions. Elimination of the catalytic nucleophile (E233A) results in an essentially inactive enzyme, consistent with the important role of this residue. However addition of small anions such as azide or formate restores activity, but as an inverting enzyme since the product formed under these conditions is the alpha-glycosyl azide. Shortening of the catalytic nucleophile (E233D) reduces the rates of both formation and hydrolysis of the glycosyl-enzyme intermediate some 3000-4000-fold. Elimination of the acid/base catalyst (E127A) yields a mutant for which the deglycosylation step is slowed some 200-300-fold as a consequence of removal of general base catalysis, but with little effect on the transition state structure at the anomeric center. Effects on the glycosylation step due to removal of the acid catalyst depend on the aglycon leaving group ability, with minimal effects on substrates requiring no general acid catalysis but large (> 10(5)-fold) effects on substrates with poor leaving groups. The Brønsted beta 1g value for hydrolysis of aryl cellobiosides was much larger (beta 1g approximately -1) for the mutant than for the wild-type enzyme (beta 1g = -0.3), consistent with removal of protonic assistance. The pH-dependence was also significantly perturbed. Mutation of a third conserved active site carboxylic acid (E123A) resulted in rate reductions of up to 1500-fold on poorer substrates, which could be largely restored by addition of azide, but without the formation of glycosyl azide products. These results suggest a simple strategy for the identification of the key active site nucleophile and acid/base catalyst residues in glycosidases without resort to active site labeling.

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Mutagenic substitution of the active-site serine residue of SGPB by either glycine or alanine has created a unique class of peptide-ligating catalysts that are useful for coupling relatively stable ester- and para-nitroanilide-activated substrates. Ligation proceeds through an acyl-enzyme intermediate involving His57. Serine to alanine mutations may provide a general strategy for converting proteases with chymotrypsin-like protein folds into peptide-coupling enzymes.

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