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P. putida mandelate racemase co-crystallized with phenylboronic acidP. putida mandelate racemase co-crystallized with phenylboronic acid
Structural highlights
FunctionPublication Abstract from PubMedBoronic 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 Ki value for PBA was 1.8 +/- 0.1 muM, and p-Cl-PBA exhibited the most potent inhibition (Ki = 81 +/- 4 nM), exceeding the binding affinity of the substrate by approximately 4 orders of magnitude. Isothermal titration calorimetric studies with the wild-type, K166M, and H297N MR variants indicated that, of the two Bronsted 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(epsilon2)-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. Potent Inhibition of Mandelate Racemase by Boronic Acids: Boron as a Mimic of a Carbon Acid Center.,Sharma AN, Grandinetti L, Johnson ER, St Maurice M, Bearne SL Biochemistry. 2020 Aug 25;59(33):3026-3037. doi: 10.1021/acs.biochem.0c00478., Epub 2020 Aug 10. PMID:32786399[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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