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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.

The replacement of the active site nucleophile Glu 358 in Agrobacterium beta-glucosidase by Asn and Gln by site-directed mutagenesis results in essentially complete inactivation of the enzyme, while replacement by Asp generates a mutant with a rate constant for the first step, formation of the glycosylenzyme, some 2500 times lower than that of the native enzyme. This low activity is shown to be a true property of the mutant and not due to contaminating wild-type enzyme by active site titration studies and also through studies of its thermal denaturation and of the pH dependence of the reaction catalyzed. Binding of ground-state inhibitors is affected relatively little by the mutation, while binding of transition-state analogues is greatly impaired, consistent with a principal role for Glu 358 being in transition-state stabilization, not substrate binding. Determination of kinetic parameters for a series of aryl glucosides revealed that the glycosylation step is rate determining for all these substrates in contrast to the native enzyme, where a switch from rate-limiting glycosylation to rate-limiting deglycosylation was observed as substrate reactivity was increased. These results coupled with secondary deuterium kinetic isotope effects of kH/kD = 1.17 and 1.12 measured for the 2,4-dinitrophenyl and p-nitrophenyl glucosides point to a principal role of the nucleophile in stabilizing the cationic transition states and in formation of the covalent intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

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Most enzymes catalyzing the hydrolysis of terminal b-N-acetylglucosaminide linkages belong to families 3 and 20 of the glycoside hydrolases ([1,2] and the glycoside hydrolases database at URL http://afmb.cnrs-mrs. fr/CAZY/). Members of the two families greatly differ in structure, enzyme mechanism, substrate specificity, and physiologic function (for a review see [3] and references cited therein). The enzymes in family 20 are designated as N-acetylhexosaminidases (EC 3.2.1.52) because they hydrolyze b-N-acetylgalactosaminides and b-N-acetylglucosaminides, with about a four-fold greater activity on the latter ([1], and references cited therein). b-N-Acetylglucosaminidases (EC 3.2.1.52) in family 3 are much more specific for the gluco-configuration, The Gram-positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular b-N-acetylglucosaminidases, a family 20 b-N-acetylhexosaminidase (Hex20), and a novel family 3-b-N-acetylglucosaminidase ⁄ b-glucosidase (Nag3), through screening of a genomic expression library, cloning of genes and analysis of their sequences. Nag3 exhibits broad substrate specificity for substituents at the C2 position of the glycone: k cat ⁄ K m values at 25°C were 0.066 s )1 AEmm )1 and 0.076 s Abbreviations DNP-2FGlc, 2¢,4¢-dinitrophenyl 2-deoxy-2-fluoro-b-D-glucopyranoside; Dp, degree of polarization;