Slow-Onset, Potent Inhibition of Mandelate Racemase by 2-Formylphenylboronic Acid. An Unexpected Adduct Clasps the Catalytic Machinery

Authors

Colin D. Douglas, Dalhousie University
Lia Grandinetti, Marquette University
Nicole M. Easton, Dalhousie University
Oliver P. Kuehm, Dalhousie University
Joshua A. Hayden, Dalhousie University
Meghan C. Hamilton, Dalhousie University
Martin St. Maurice, Marquette UniversityFollow
Stephen L. Bearne, Dalhousie University

Document Type

Article

Publication Date

8-2021

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Abstract

o-Carbonyl arylboronic acids such as 2-formylphenylboronic acid (2-FPBA) are employed in biocompatible conjugation reactions with the resulting iminoboronate adduct stabilized by an intramolecular N–B interaction. However, few studies have utilized these reagents as active site-directed enzyme inhibitors. We show that 2-FPBA is a potent reversible, slow-onset inhibitor of mandelate racemase (MR), an enzyme that has served as a valuable paradigm for understanding enzyme-catalyzed abstraction of an α-proton from a carbon acid substrate with a high pK_a. Kinetic analysis of the progress curves for the slow onset of inhibition of wild-type MR using a two-step kinetic mechanism gave K_i and K_i* values of 5.1 ± 1.8 and 0.26 ± 0.08 μM, respectively. Hence, wild-type MR binds 2-FPBA with an affinity that exceeds that for the substrate by ∼3000-fold. K164R MR was inhibited by 2-FPBA, while K166R MR was not inhibited, indicating that Lys 166 was essential for inhibition. Unexpectedly, mass spectrometric analysis of the NaCNBH₃-treated enzyme–inhibitor complex did not yield evidence of an iminoboronate adduct. ¹¹B nuclear magnetic resonance spectroscopy of the MR·2-FPBA complex indicated that the boron atom was sp³-hybridized (δ 6.0), consistent with dative bond formation. Surprisingly, X-ray crystallography revealed the formation of an N^ζ–B dative bond between Lys 166 and 2-FPBA with intramolecular cyclization to form a benzoxaborole, rather than the expected iminoboronate. Thus, when o-carbonyl arylboronic acid reagents are employed to modify proteins, the structure of the resulting product depends on the protein architecture at the site of modification.

Comments

Accepted version. Biochemistry, Vol. 60, No. 32 (August 2021): 2508-2518. DOI. © 2021 American Chemical Society. Used with permission.

Recommended Citation

Douglas, Colin D.; Grandinetti, Lia; Easton, Nicole M.; Kuehm, Oliver P.; Hayden, Joshua A.; Hamilton, Meghan C.; St. Maurice, Martin; and Bearne, Stephen L., "Slow-Onset, Potent Inhibition of Mandelate Racemase by 2-Formylphenylboronic Acid. An Unexpected Adduct Clasps the Catalytic Machinery" (2021). Biological Sciences Faculty Research and Publications. 882.
https://epublications.marquette.edu/bio_fac/882