Potent Inhibition of Mandelate Racemase by Boronic Acids: Boron as a Mimic of a Carbon Acid Center

Authors

Amar Nath Sharma, Dalhousie University
Lia Grandinetti, Marquette University
Erin R. Johnson, Dalhousie University
Martin St. Maurice, Marquette UniversityFollow
Stephen L. Bearne, Dalhousie University

Document Type

Article

Language

eng

Publication Date

7-2020

Publisher

American Chemical Society Publications

Source Publication

Biochemistry

Source ISSN

0006-2960

Abstract

Boronic acids have been successfully employed as inhibitors of hydrolytic enzymes. Typically, an enzymatic nucleophile catalyzing hydrolysis adds to the electrophilic boron atom forming a tetrahedral species that mimics the intermediate(s)/transition state(s) for the hydrolysis reaction. We show that para-substituted phenylboronic acids (PBAs) are potent competitive inhibitors of mandelate racemase (MR), an enzyme that catalyzes a 1,1-proton transfer rather than a hydrolysis reaction. The K_i value for PBA was 1.8 ± 0.1 μM, and p-Cl-PBA exhibited the most potent inhibition (K_i = 81 ± 4 nM), exceeding the binding affinity of the substrate by ∼4 orders of magnitude. Isothermal titration calorimetric studies with the wild-type, K166M, and H297N MR variants indicated that, of the two Brønsted acid–base catalysts Lys 166 and His 297, the former made the greater contribution to inhibitor binding. The X-ray crystal structure of the MR·PBA complex revealed the presence of multiple H-bonds between the boronic acid hydroxyl groups and the side chains of active site residues, as well as formation of a His 297 N^ε2–B dative bond. The dramatic upfield change in chemical shift of 27.2 ppm in the solution-phase ¹¹B nuclear magnetic resonance spectrum accompanying binding of PBA by MR was consistent with an sp³-hybridized boron, which was also supported by density-functional theory calculations. These unprecedented findings suggest that, beyond substituting boron at carbon centers participating in hydrolysis reactions, substitution of boron at the acidic carbon center of a substrate furnishes a new approach for generating inhibitors of enzymes catalyzing the deprotonation of carbon acid substrates.

Comments

Accepted version. Biochemistry, Vol. 59, No. 33 (July 2020): 3026-3037. DOI. © 2020 American Chemical Society Publications. Used with permission.

Recommended Citation

Sharma, Amar Nath; Grandinetti, Lia; Johnson, Erin R.; St. Maurice, Martin; and Bearne, Stephen L., "Potent Inhibition of Mandelate Racemase by Boronic Acids: Boron as a Mimic of a Carbon Acid Center" (2020). Biological Sciences Faculty Research and Publications. 824.
https://epublications.marquette.edu/bio_fac/824