Dissecting the Electrostatic Interactions and pH-Dependent Activity of a Family 11 Glycosidase †,‡ Manish D. Joshi, §,| Gary Sidhu, § Jens E. Nielsen, ⊥ Gary D. Brayer, § Stephen G. Withers, §,# and Lawrence P. McIntosh *,§,#,O Department of Biochemistry and Molecular Biology, Department of Chemistry, and The Biotechnology Laboratory, UniVersity of British Columbia, VancouVer, British Columbia, Canada V6T 1Z3, and European Molecular Biology Laboratory, Heidelberg, Germany 69117 ReceiVed March 15, 2001; ReVised Manuscript ReceiVed June 19, 2001 ABSTRACT: Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (Glu78, pK a 4.6) and the acid/base catalyst (Glu172, pK a 6.7) gives rise to its pH-dependent activity profile. Inspection of the crystal structure of BCX reveals that Glu78 and Glu172 are in very similar environments and are surrounded by several chemically equivalent and highly conserved active site residues. Hence, there are no obvious reasons why their apparent pK a values are different. To address this question, a mutagenic approach was implemented to determine what features establish the pK a values (measured directly by 13 C NMR and indirectly by pH-dependent activity profiles) of these two catalytic carboxylic acids. Analysis of several BCX variants indicates that the ionized form of Glu78 is preferentially stabilized over that of Glu172 in part by stronger hydrogen bonds contributed by two well-ordered residues, namely, Tyr69 and Gln127. In addition, theoretical pK a calculations show that Glu78 has a lower pK a value than Glu172 due to a smaller desolvation energy and more favorable background interactions with permanent partial charges and ionizable groups within the protein. The pK a value of Glu172 is in turn elevated due to electrostatic repulsion from the negatively charged glutamate at position 78. The results also indicate that all of the conserved active site residues act concertedly in establishing the pK a values of Glu78 and Glu172, with no particular residue being singly more important than any of the others. In general, residues that contribute positive charges and hydrogen bonds serve to lower the pK a values of Glu78 and Glu172. The degree to which a hydrogen bond lowers a pK a value is largely dependent on the length of the hydrogen bond (shorter bonds lower pK a values more) and the chemical nature of the donor (COOH > OH > CONH 2 ). In contrast, neighboring carboxyl groups can either lower or raise the pK a values of the catalytic glutamic acids depending upon the electrostatic linkage of the ionization constants 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 residues within the active site, activity is usually compromised due to the loss of important ground and/or transition state interactions. These results suggest that the pH optima of an enzyme might be best engineered by making strategic amino acid substitutions, at positions outside of the “core” active site, that electrostatically influence catalytic residues without perturbing their immediate structural environment. Enzymes catalyze virtually all biochemical reactions in a bewildering array of organisms and often under extremes of environmental conditions. Remarkably, this can be carried out using a rather limited repertoire of amino acids that serve as nucleophiles, electrophiles, and general acids and bases. Clearly, enzyme structures have evolved in part to modulate the physiochemical properties of these amino acids, as required for catalysis of a particular reaction under a given set of conditions. Since these catalytic amino acids generally have ionizable side chains, one critical property is their precise pK a value within the context of the native enzyme. † This work was funded by the Government of Canada’s Network of Centres of Excellence Program supported by the Medical Research Council (MRC) and the Natural Sciences and Engineering Research Council (NSERC) through the Protein Engineering Network of Centres of Excellence (PENCE Inc.). L.P.M. acknowledges the Alexander von Humbolt Foundation for support of a sabbatical leave with Dr. M. Nilges at The European Molecular Biology Laboratory, Heidelberg, Germany, and the Candian Institutes of Health Research for a Scientist Award. ‡ Coordinates for the structures described in this work have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) (accession numbers 1HV0 and 1HV1). * Address correspondence to this author at the Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3. Tel: (604) 822-3341. Fax: (604) 822-5227. E-mail: mcintosh@ otter.biochem.ubc.ca. § Department of Biochemistry and Molecular Biology, University of British Columbia. | Present address: Ontario Cancer Institute/Princess Margaret Hos- pital, Division of Molecular and Structural Biology, University of Toronto, Department of Medical Biophysics, Toronto, ON, Canada M5G 2M9. ⊥ European Molecular Biology Laboratory. # Department of Chemistry, University of British Columbia. O The Biotechnology Laboratory, University of British Columbia. 10115 Biochemistry 2001, 40, 10115-10139 10.1021/bi0105429 CCC: $20.00 © 2001 American Chemical Society Published on Web 08/07/2001