@article {3762,
	title = {The pK(a) of the general acid/base carboxyl group of a glycosidase cycles during catalysis: A C-13-NMR study of Bacillus circuluns xylanase},
	journal = {Biochemistry},
	volume = {35},
	number = {31},
	year = {1996},
	note = {ISI Document Delivery No.: VB211Times Cited: 149Cited Reference Count: 43},
	month = {Aug},
	pages = {9958-9966},
	type = {Article},
	abstract = {The 20 kDa xylanase from Bacillus circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl-enzyme intermediate. In this double-displacement reaction, Glu78 functions as a nucleophile to form the intermediate, while Glu172 acts as a general acid catalyst during glycosylation, protonating the departing aglycone, and then as a general base during deglycosylation, deprotonating the attacking water, The dual role of Glu172 places specific demands upon its ionization states and hence pK(a) values. C-13-NMR titrations of xylanase, labeled with [delta-C-13]glutamic acid, have revealed pK(a) values of 4.6 and 6.7 for Glu78 and Glu172, respectively. These agree well with the apparent pK(a) values obtained from a study of the pH dependence of k(cat)/K-m and demonstrate that, at the enzyme{\textquoteright}s pH optimum of 5.7, the nucleophile Glu78 is deprotonated and the general acid Glu172 initially protonated. Remarkably, the pK(a) for Glu172 drops to 4.2 in a trapped covalent glycosyl-enzyme intermediate, formed by reaction with 2{\textquoteright},4{\textquoteright}-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside [Miao et al. (1994) Biochemistry 33, 7027-7032], A similar pK(a) is measured for Glu172 when a glutamine is present at position 78. This large decrease in pK(a) of similar to 2.5 units is consistent with the role of Glu172 as a general base catalyst in the deglycosylation step and appears to be a consequence of both reduced electrostatic repulsion due to neutralization of Glu78 and a conformational change in the protein. Such {\textquoteright}{\textquoteright}pK(a) cycling{\textquoteright}{\textquoteright} during catalysis is likely to be a common phenomenon in glycosidases.},
	keywords = {ACTIVE-SITE RESIDUES, AMINO-ACIDS, beta-glucosidase, ELECTROSTATIC INTERACTIONS, ESCHERICHIA-COLI, IDENTIFICATION, LYSOZYME, MECHANISMS, NUCLEAR-MAGNETIC-RESONANCE, PROTEINS},
	isbn = {0006-2960},
	url = {<Go to ISI>://A1996VB21100003},
	author = {McIntosh, L. P. and Hand, G. and Johnson, P. E. and Joshi, M. D. and Korner, M. and Plesniak, L. A. and Ziser, L. and Wakarchuk, W. W. and Withers, S. G.}
}
