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

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 11 B nuclear magnetic resonance spectrum accompanying binding of PBA by MR was consistent with an sp 3 -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.

Structural insights into the catalytic and inhibitory mechanisms of the flavin transferase FmnB in Listeria monocytogenes

“…This action is similar to the formation of oxyanion holes in the triplet catalysis of subtilisin, which is stabilized by Asn, Thr, and the backbone NH of Ser. 30 Consistent with our enzymatic activity assays, substituting these residues with alanine resulted in significant or complete loss of enzymatic activity (Figure 3G ). Notably, substituting Ser257 with alanine partially reduced the enzymatic activity.…”

“…Meanwhile, Asp301 stabilizes the oxygen in the transition state through the coordination of magnesium ions, while Asn216 stabilizes Asp301 through a salt bridge with Asp301 (Figures 3D and S13C). This action is similar to the formation of oxyanion holes in the triplet catalysis of subtilisin, which is stabilized by Asn, Thr, and the backbone NH of Ser 30 . Consistent with our enzymatic activity assays, substituting these residues with alanine resulted in significant or complete loss of enzymatic activity (Figure 3G).…”

Combining flavin photocatalysis with parallel synthesis: a general platform to optimize peptides with non-proteinogenic amino acids

“…Moreover, radicals are known to modify histidine, tyrosine, and tryptophan amino acids. 36 And nally, nucleophilic residues including histidine, 37,38 serine (S), 39 and lysine (K) 40,41 are known to coordinate to boronic acids. Subjecting the 11-mer peptide to our optimized reaction conditions afforded 50% of a mono-labeled product in the presence of (8-methoxy-2methylquinolin-5-yl)boronic acid 6B (Scheme 1C).…”

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

“…°C, and at various times (2,7,11,16,20,24,29,44, and 60 min), an aliquot was removed and diluted 100-fold into assay buffer containing (S)-mandelate (final concentration of 2.0 mM) and the steady state velocity was then measured using the standard MR assay.…”

“…Consequently, we rationalized that 2-FPBA should be bound with a high affinity as we recently demonstrated for phenylboronic acid (PBA) 29 and form a stabilized iminoboronate through Schiff base formation with one of the Lys residues present in the KXK motif. Herein, we show that 2-FPBA is the first reversible slow-onset inhibitor of MR, binding with an affinity that exceeds that of PBA.…”