Identification of Tyr241 as a key catalytic base in the Family 4 glycoside hydrolase BglT from Thermotoga maritima.

While the vast majority of glycosidases catalyze glycoside hydrolysis via oxocarbenium ion-like transition states and typically employ carboxylic acid residues as acid/base or nucleophile catalysts, two sub-families of these enzymes (GH4 and GH109 in the CAZY classification) carry out hydrolysis via a redox-assisted mechanism involving anionic transition states. While good evidence has been obtained for this mechanism, the identities of the catalytic residues involved have not yet been confirmed. Mechanistic analyses of mutants of the 6-phospho-β-glucosidase from Thermatoga maritima (BglT), in which the active site tyrosine residue (Tyr 241) has been replaced by Phe and Ala, provide strong support for its role as a catalytic base. The pH-dependence of kcat and kcat/KM, particularly of the acidic limb corresponding to the base, is shifted relative to that of the wild-type enzyme. Kinetic isotope effects for hydrolysis of substrates deuterated at C-1, C-2 and C-3 by the Tyr 241 mutants are strongly pH-dependent, with essentially full primary kinetic isotope effects being observed for the 2-deutero substrate at low pH with the Tyr241Ala mutant. This is consistent with a slowing of the deprotonation step upon removal of the base.