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Biochem J. 1998 Feb 15;330 ( Pt 1):203-9.

Mechanistic consequences of replacing the active-site nucleophile Glu-358 in Agrobacterium sp. beta-glucosidase with a cysteine residue.

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  • 1Protein Engineering Network of Centres of Excellence and the Departments of Chemistry and Microbiology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.


Retaining glycosidases achieve the hydrolysis of glycosidic bonds through the assistance of two key active-site carboxyls. One carboxyl functions as a nucleophile/leaving group, and the other acts as the acid-base catalyst. It has been suggested that a cysteine residue could fulfil the role of the active site nucleophile [Hardy and Poteete (1991) Biochemistry 30, 9457-9463]. To test the validity of this proposal, a kinetic evaluation was conducted on the active-site nucleophile cysteine mutant (Glu-358-->Cys) of the retaining beta-glucosidase from Agrobacterium sp. The Glu-358-->Cys mutant was able to complete the first step (glycosylation) of the enzymic mechanism, forming a covalent glycosyl-enzyme intermediate, but the rate constant for this step was decreased to 1/10(6) of that of the native enzyme. The subsequent hydrolysis (deglycosylation) step was also severely affected by the replacement of Glu-358 with a cysteine residue, with the rate constant being depressed to 1/10(7) or less. Thus Cys-358 functions inefficiently in both the capacity of catalytic nucleophile and leaving group. On the basis of these results it seems unlikely that the role of the active-site nucleophile in retaining glycosidases could successfully be filled by a cysteine residue.

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