Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase

Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (GIu78, pK(a) 4.6) and the acid/base catalyst (Glu 172, pK(a) 6.7) gives rise to its pH-d ependent activity profile. Inspection of the crystal structure of BCX revea ls that Glu78 and Glu172 are in very similar environments and are surrounde d by several chemically equivalent and highly conserved active site residue s. Hence, there are no obvious reasons why their apparent pKa values are di fferent. To address this question, a mutagenic approach was implemented to determine what features establish the pKa values (measured directly by C-13 NMR and indirectly by pH-dependent activity profiles) of these two catalyt ic carboxylic acids. Analysis of several BCX variants indicates that the io nized form of Glu78 is preferentially stabilized over that of Glu 172 in pa rt by stronger hydrogen bonds contributed by two well-ordered residues, nam ely, Tyr69 and Gln127. In addition, theoretical pKa calculations show that Glu78 has a lower pKa value than Glu 172 due to a smaller desolvation energ y and more favorable background interactions with permanent partial charges and ionizable groups within the protein. The pKa value of Glu172 is in tur n elevated due to electrostatic repulsion from the negatively charged gluta mate at position 78. The results also indicate that all of the conserved ac tive site residues act concertedly in establishing the pKa values of Glu78 and Glu 172, with no particular residue being singly more important than an y of the others. In general, residues that contribute positive charges and hydrogen bonds serve to lower the pKa values of Glu78 and Glu172. The degre e to which a hydrogen bond lowers a pKa value is largely dependent on the l ength of the hydrogen bond (shorter bonds lower pKa values more) and the ch emical nature of the donor (COOH > OH > CONH2). In contrast, neighboring ca rboxyl groups can either lower or raise the pKa values of the catalytic glu tamic acids depending upon the electrostatic linkage of the ionization cons tants of the residues involved in the interaction. While the pH optimum of BCX can be shifted from -1.1 to +0.6 pH units by mutating neighboring resid ues within the active site, activity is usually compromised due to the loss of important ground and/or transition state interactions. These results su ggest that the pH optima of an enzyme might be best engineered by making st rategic amino acid substitutions, at positions outside of the "core" active site, that electrostatically influence catalytic residues without perturbi ng their immediate structural environment.