@article { ISI:000072441800030,
	title = {Mechanistic consequences of replacing the active-site nucleophile Glu-358 in Agrobacterium sp. beta-glucosidase with a cysteine residue},
	journal = {BIOCHEMICAL JOURNAL},
	volume = {330},
	number = {1},
	year = {1998},
	month = {FEB 15},
	pages = {203-209},
	abstract = {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 {\textendash}> Cys) of the retaining beta-glucosidase from Agrobacterium sp. The Glu-358 {\textendash}> Cys mutant was able to complete the first step (glycosylation) of the enzymic mechanism, forming a covalent glycosylenzyme 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.},
	issn = {0264-6021},
	author = {Lawson, SL and WARREN, RAJ and Withers, SG}
}
