@article { ISI:A1995TD84500034,
	title = {IDENTIFICATION OF THE ACID/BASE CATALYST IN AGROBACTERIUM-FAECALIS BETA-GLUCOSIDASE BY KINETIC-ANALYSIS OF MUTANTS},
	journal = {BIOCHEMISTRY},
	volume = {34},
	number = {44},
	year = {1995},
	month = {NOV 7},
	pages = {14554-14562},
	abstract = {The catalytic mechanism of the retaining beta-glucosidase (Abg) from Agrobacterium faecalis involves a double-displacement process in which an alpha-glucosyl-enzyme intermediate is formed with general acid catalytic assistance and then hydrolyzed with general base assistance. Glu170 was identified as an important residue, possibly the acid/base catalyst, on the basis of sequence alignments. This glutamate is conserved in almost all enzymes in family 1 of glycoside hydrolases. Detailed pre-steady-state and steady-state kinetic analyses of the mutant E170G suggested very strongly that Glu170 is the acid/base catalyst. First, k(cat) values were invariant with pH over the range of 5.0-9.0. Secondly, rates of formation of the glycosyl-enzyme, calculated from k(cat)/K-m and k(2), were similar to those of wild-type enzyme for substrates not requiring protonic assistance but dramatically reduced for those needing acid catalysis. Thirdly, addition of azide as a competitive nucleophile increased k(cat) values 100-300-fold for substrates whose rate-limiting step is deglycosylation, yielding beta-glucosyl azide, but had no effect on the wild-type enzyme. Other anionic nucleophiles had similar, but less dramatic effects. Previous results {[}Gebler, J. C., et al. (1995) 34, 14547-14553] had indicated that Tyr298 is important for catalysis. The kinetic consequences of the mutations in the double mutant E170G-Y298F are additive, resulting in a 10(6)-fold reduction in k(cat) values and allowing the accumulation of a stable (t(1/2) > 7 h) glucosyl-enzyme intermediate. Thus, Glu170 and Tyr298 function independently, and a possible role for Tyr298 in modulating the pK(2) of the catalytic nucleophile is proposed.},
	issn = {0006-2960},
	doi = {10.1021/bi00044a034},
	author = {WANG, Q and TRIMBUR, D and GRAHAM, R and WARREN, RAJ and Withers, SG}
}
