Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism.
Biochemistry
; 57(23): 3244-3251, 2018 06 12.
Article
en En
| MEDLINE
| ID: mdl-29489337
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 reported and revealed sulfite binding to the pyridinium C4 atom of the reduced pincer cofactor, mimicking hydride reduction during a PCHT catalytic cycle. Finally, computational modeling supports hydride transfer to the cofactor at the C4 position or to the nickel atom, but with formation of a nickel-hydride species requiring dissociation of the His200 metal ligand. In aggregate, these studies provide compelling evidence that the nickel-pincer cofactor acts by a PCHT mechanism.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Compuestos Organometálicos
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Protones
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Proteínas Bacterianas
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Coenzimas
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Racemasas y Epimerasas
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Lactobacillus plantarum
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Níquel
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Biochemistry
Año:
2018
Tipo del documento:
Article
Pais de publicación:
Estados Unidos