Modulating the Nucleophile of a Glycoside Hydrolase through Site- Specific Incorporation of Fluoroglutamic Acids Miriam P. Kö tzler, † Kyle Robinson, † Hong-Ming Chen, † Mark Okon, †,‡ Lawrence P. McIntosh, †,‡,§ and Stephen G. Withers* ,†,‡ † Department of Chemistry, ‡ Department of Biochemistry and Molecular Biology, and § Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada * S Supporting Information ABSTRACT: Understanding the detailed mechanisms of enzyme-catalyzed hydrolysis of the glycosidic bond is fundamentally important, not only to the design of tailored cost-efficient, stable and specific catalysts but also to the development of specific glycosidase inhibitors as therapeutics. Retaining glycosidases employ two key carboxylic acid residues, typically glutamic acids, in a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. One Glu functions as a nucleophile while the other acts as a general acid/base. A significant part of enzymatic proficiency is attributed to a “perfect match” of the electrostatics provided by these key residues, a hypothesis that has been remarkably difficult to prove in model systems or in enzymes themselves. We experimentally probe this synergy by preparing synthetic variants of a model glycosidase Bacillus circulans β-xylanase (Bcx) with the nucleophile Glu78 substituted by 4-fluoro or 4,4-difluoroglutamic acid to progressively reduce nucleophilicity. These Bcx variants were semisynthesized by preparation of optically pure fluoroglutamic acid building blocks, incorporation into synthetic peptides, and ligation onto a truncated circular permutant of Bcx. By measuring the effect of altered electrostatics in the active site on enzyme kinetic constants, we show that lowering the nucleophile pKa by two units shits the pH-dependent activity by one pH unit. Linear free energy correlations using substrates of varying leaving group ability indicate that by reducing nucleophilic catalysis the concerted mechanism of the enzyme is disrupted and shifted toward a dissociative pathway. Our study represents the first example of site-specific introduction of fluorinated glutamic acids into any protein. Furthermore, it provides unique insights into the synergy of nucleophilic and acid/base catalysis within an enzyme active site. ■ INTRODUCTION Glycoside hydrolases are among the most proficient catalysts known, accelerating reactions by up to ∼10 17 fold above the sluggish, uncatalyzed processes. 1 They are also among the most widely used enzymes commercially, with applications ranging from food processing and brewing through to the textiles, biofuel, and pulp and paper industries. 2-5 Some of these enzymes, among them the Bacillus circulans endo-β-1,4- xylanase (Bcx), have been engineered to optimize their stability and activity for use under harsh processing conditions. 4,6,7 Further fine tuning of catalytic properties, including shifting or broadening pH-dependent activity profiles, would be of great interest. However, this requires a deep mechanistic understanding of factors underlying catalysis, such as protonation equilibria in the active site and electrostatic stabilization of the transition states. The general models of catalytic mechanisms employed by glycoside hydrolases were introduced by Koshland over half a century ago 8 and have been heavily refined in the interim. 9,10 Inverting glycoside hydrolases (which effect inversion of anomeric stereochemistry) employ a direct displacement mechanism via a single oxocarbenium-like transition state. By contrast, retaining glycoside hydrolases use a two-step mechanism involving two oxocarbenium-like transition states (ii and iv in Scheme 1). One carboxylate (Glu78 in Bcx) acts as a nucleophilic catalyst, attacking the anomeric carbon to form a covalent glycosyl enzyme intermediate (glycosylation step i to ii). This transient covalent intermediate (iii) is then hydrolyzed in the second step, releasing the free reducing sugar with net retention of anomeric configuration (deglycosylation Received: April 20, 2018 Published: June 12, 2018 Article pubs.acs.org/JACS Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX-XXX © XXXX American Chemical Society A DOI: 10.1021/jacs.8b04235 J. Am. Chem. Soc. XXXX, XXX, XXX-XXX Downloaded via UNIV OF BRITISH COLUMBIA on June 28, 2018 at 17:54:08 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.