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

o-Carbonyl arylboronic acids such as 2-formylphe-nylboronic 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 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 mu M, respectively. Hence, wild-type MR binds 2-FPBA with an affinity that exceeds that for the substrate by similar to 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. B-11 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.

Automated summary

2-FPBA is shown to be a potent reversible, slow-onset inhibitor of mandelate racemase (MR), forming a benzoxaborole through intramolecular cyclization due to the N-zeta-B dative bond formation between Lys 166 and 2-FPBA. The structure of the resulting product when using o-carbonyl arylboronic acid reagents to modify proteins depends on the protein architecture at the site of modification.