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Publication Abstract from PubMed

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 alpha-proton from a carbon acid substrate with a high pKa. Kinetic analysis of the progress curves for the slow onset of inhibition of wild-type MR using a two-step kinetic mechanism gave Ki and Ki* values of 5.1 +/- 1.8 and 0.26 +/- 0.08 muM, respectively. Hence, wild-type MR binds 2-FPBA with an affinity that exceeds that for the substrate by approximately 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. (11)B nuclear magnetic resonance spectroscopy of the MR.2-FPBA complex indicated that the boron atom was sp(3)-hybridized (delta 6.0), consistent with dative bond formation. Surprisingly, X-ray crystallography revealed the formation of an N(zeta)-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.

Slow-Onset, Potent Inhibition of Mandelate Racemase by 2-Formylphenylboronic Acid. An Unexpected Adduct Clasps the Catalytic Machinery.,Douglas CD, Grandinetti L, Easton NM, Kuehm OP, Hayden JA, Hamilton MC, St Maurice M, Bearne SL Biochemistry. 2021 Aug 2. doi: 10.1021/acs.biochem.1c00374. PMID:34339165^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.