Analyses of pre-steady-state kinetics and isotope effects of the γ-elimination reaction catalyzed by Citrobacter freundii methionine γ-lyase.

Autor: Kuznetsova AA; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk, 630090, Russia., Faleev NG; Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow, 119991, Russia., Morozova EA; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia., Anufrieva NV; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia., Gogoleva OI; Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow, 119991, Russia., Tsvetikova MA; Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow, 119991, Russia., Fedorova OS; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk, 630090, Russia., Demidkina TV; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia. Electronic address: tvdemidkina@yandex.ru., Kuznetsov NA; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk, 630090, Russia. Electronic address: nikita.kuznetsov@niboch.nsc.ru.
Jazyk: angličtina
Zdroj: Biochimie [Biochimie] 2022 Oct; Vol. 201, pp. 157-167. Date of Electronic Publication: 2022 Jun 09.
DOI: 10.1016/j.biochi.2022.06.002
Abstrakt: Methionine γ-lyase (MGL) is a pyridoxal 5'-phosphate-dependent enzyme catalyzing γ-elimination in l-methionine. Pyridoxal 5'-phosphate-dependent enzymes have unique spectral properties that allow to monitor sequential formation and decomposition of various intermediates via the detection of absorbance changes. The kinetic mechanism of the γ-elimination reaction catalyzed by Citrobacter freundii MGL was elucidated here by fast stopped-flow kinetic analysis. Single-wavelength detection of characteristic absorbance changes enabled us to compare transformations of intermediates in the course of the reaction with different substrates. The influence of various γ-substituents in the substrate on the formation of key intermediates was estimated. Kinetic isotope effects of α- and β-protons were determined using deuterium-substituted l-methionine. Contributions of amino acid residues Tyr113 and Tyr58 located in the active site on the formation and decomposition of reaction intermediates were identified too. α-Aminocrotonate formation is the rate-limiting step of the enzymatic γ-elimination reaction. Kinetic isotope effects strongly support concerted reaction mechanisms of transformation between an external aldimine and a ketimine intermediate as well as a ketimine intermediate and an unsaturated ketimine.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)
Databáze: MEDLINE
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