Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism
| Autor: | Joel A Rankin, Matthias Fellner, John McCracken, Sergey A. Varganov, Robert C. Mauban, Robert P. Hausinger, Benoît Desguin, Jian Hu |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Stereochemistry Coenzymes Racemases and Epimerases chemistry.chemical_element Isomerase Crystallography X-Ray 010402 general chemistry 01 natural sciences Biochemistry Cofactor 03 medical and health sciences Electron transfer Bacterial Proteins Protein Domains Nickel Lactate racemase Organometallic Compounds Pincer ligand biology Hydride Electron Spin Resonance Spectroscopy 0104 chemical sciences 030104 developmental biology chemistry biology.protein Spectrophotometry Ultraviolet Protons Lactobacillus plantarum Lactate racemization |
| Zdroj: | Biochemistry. 57:3244-3251 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.8b00100 |
| Popis: | 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 ... |
| Databáze: | OpenAIRE |
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