Modulating the Nucleophile of a Glycoside Hydrolase through Site-Specific Incorporation of Fluoroglutamic Acids

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 p Ka 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.

Strawberry Mottle Virus (Family Secoviridae , Order Picornavirales ) Encodes a Novel Glutamic Protease To Process the RNA2 Polyprotein at Two Cleavage Sites

“…However, local pKa effects conferred by surrounding amino acid residues can alter the charge of catalytic aspartic or glutamic acids under physiological pH, as shown for the dimeric human immunodeficiency virus-1 (HIV-1) aspartic protease (41)(42)(43)(44)(45). Similarly, the activity of glycoside hydrolases with concanavalin A-like lectin/glucanase folds at physiological pH was attributed to local pKa effects at the two catalytic glutamic acid residues (46,47). In the case of the HIV-1 protease, binding to substrates can also contribute to alterations of the pKa values of the catalytic aspartic acid residues (43).…”

A Structural Dissection of the Active Site of the Lytic Transglycosylase MltE from Escherichia coli

“…Their transacetalization reaction is mediated by a pivotal acidic catalytic residue, wherein the complementary motions of the substrate and the protein in the catalytic cycle direct an intramolecular displacement at the glycosidic carbon of the MurNAc. In other GH families, catalysis occurs through transition states wherein local charge development at this glycosidic carbon is suppressed, as a consequence of matched nucleophilicity and nucleofugacity . In this mechanism a discrete oxocarbenium species is not encountered, and the bond-forming and bond-cleaving processes at the glycosidic carbon are near-concerted.…”

Comprehensive strategies of Lignocellulolytic enzyme production from microbes and their applications in various commercial-scale faculties

“…Cellulase digests cellulose, and xylanase digests hemicellulose of the plants. The small amount of pre-digested feedstock is enough to rectify the whole day's diet (Miriam et al, 2018;Murad & Azzaz, 2010). Semi-digestive feed, which contains a lignocellulolytic enzyme, can reduce the burden of the animal digestive system and time consumed for digestion and adsorption (Christina et al, 2015).…”

“…It is possible to increase systematic biofuel production by focusing on the thermostability control of respective enzymes. Enzyme activity depends on the intramolecular interaction of the protein fold (Kötzler et al, 2018) . Enzymes in endoglucanase [EC 3.2.1.4] and exoglucanase [EC 3.2.1.91] families actively degrade the plant biomass into cellobiose and celloclextrims.…”