Hydrogen bonding and catalysis: A novel explanation for how a single aminoacid substitution can change the pH optimum of a glycosidase

The pH optima of family 11 xylanases are well correlated with the nature of the residue adjacent to the acid/base catalyst. In xylanases that function optimally under acidic conditions, this residue is aspartic acid, whereas it is asparagine in those that function under more alkaline conditions. Pre vious studies of wild-type (WT) Bacillus circulans xylanase (BCX), with an asparagine residue at position 35, demonstrated that its pH-dependent activ ity follows the ionization states of the nucleophile Glu78 (pK(a) 4.6) and the acid/base catalyst Glu172 (pK(a) 6.7). As predicted from sequence compa risons, substitution of this asparagine residue with an aspartic acid resid ue (N35D BCX) shifts its pH optimum from 5.7 to 4.6, with an similar to 20 % increase in activity. The bell-shaped pH-activity profile of this mutant enzyme follows apparent pK(a) values of 3.5 and 5.8. Based on C-13-NMR titr ations, the predominant pK(a) values of its active-site carboxyl groups are 3.7 (Asp35), 5.7 (Glu78) and 8.4 (Glu172). Thus, in contrast to the WT enz yme, the pH-activity profile of N35D BCX appears to be set by Asp35 and Glu 78. Mutational, kinetic, and structural studies of N35D BCX, both in its na tive and covalently modified 2-fluoro-xylobiosyl glycosyl-enzyme intermedia te states, reveal that the xylanase still follows a double-displacement mec hanism with Glu78 serving as the nucleophile. We therefore propose that Asp 35 and Glu172 function together as the general acid/base catalyst, and that N35D BCX exhibits a "reverse protonation" mechanism in which it is catalyt ically active when Asp35, with the lower pK(a), is protonated, while Glu78, with the higher pK(a), is deprotonated. This implies that the mutant enzym e must have an inherent catalytic efficiency at least 100-fold higher than that of the parental WT, because only similar to 1% of its population is in the correct ionization state for catalysis at its pH optimum. The increase d efficiency of N35D BCX, and by inference all "acidic" family 11 xylanases , is attributed to the formation of a short (2.7 Angstrom) hydrogen bond be tween Asp35 and Glu172, observed in the crystal structure of the glycosyl-e nzyme intermediate of this enzyme, that will substantially stabilize the tr ansition state for glycosyl transfer. Such a mechanism may be much more com monly employed than is generally realized, necessitating careful analysis o f the pH-dependence of enzymatic catalysis. (C) 2000 Academic Press.