Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism

Biochemistry
Joel A RankinRobert P Hausinger

Abstract

Lactate racemase (LarA) of Lactobacillus plantarum contains a novel organometallic cofactor with nickel coordinated to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide, but its function in the enzyme mechanism has not been elucidated. This study presents direct evidence that the nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization. No signal was detected by electron paramagnetic resonance spectroscopy for LarA in the absence or presence of substrate, consistent with a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Pyruvate, the predicted intermediate for a PCHT mechanism, was observed in quenched solutions of LarA. A normal substrate kinetic isotope effect ( kH/ kD of 3.11 ± 0.17) was established using 2-α-2H-lactate, further supporting a PCHT mechanism. UV-visible spectroscopy revealed a lactate-induced perturbation of the cofactor spectrum, notably increasing the absorbance at 340 nm, and demonstrated an interaction of the cofactor with the inhibitor sulfite. A crystal structure of LarA provided greater resolution (2.4 Å) than previously r...Continue Reading

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Citations

Oct 20, 2020·ChemCatChem·Jorge NevarezRobert P Hausinger
Oct 24, 2020·Scientific Reports·Benoît DesguinPatrice Soumillion
Feb 4, 2019·Current Opinion in Structural Biology·Robert P Hausinger
Feb 1, 2019·Journal of Chemical Information and Modeling·Edoardo Jun MattioliMatteo Calvaresi

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