Kinetic mechanism of a recombinant Arabidopsis glyoxylate reductase: studies of initial velocity, dead-end inhibition and product inhibition.

Kinetic analysis of substrate specificity revealed that a recombinant Arabidopsis protein catalyzes the conversion of glyoxylate to glycolate (Km,glyoxylate = 4.5 μmol·L^–1) and succinic semialdehyde (SSA) to γ-hydroxybutyrate (Km, SSA = 0.87 mmol·L^–1) via an essentially irreversible, NADPH-based mechanism. In this report, the enzyme was further characterized via initial-velocity, dead-end inhibition and product inhibition studies. The kinetic mechanism was ordered Bi Bi, involving the complexation of NADPH to the enzyme before glyoxylate or SSA, and the release of NADP⁺ before glycolate or γ-hydroxybutyrate, respectively. It can be concluded that the enzyme functions as a NADPH-dependent glyoxylate reductase (EC 1.1.1.79) or possibly an aldehyde reductase (EC 1.1.1.2), and the kinetic mechanism involved is consistent with that found in members of both the aldo-keto reductase and 3-hydroxyisobutyrate dehydrogenase-related superfamilies of enzymes. Since NADP⁺ was an effective competitive inhibitor with respect to NADPH (Ki = 1–3 µmol·L^–1), it is proposed that the ratio of NADPH/NADP⁺ regulates enzymatic activity in planta. [ABSTRACT FROM AUTHOR]

L’analyse cinétique de la spécificité du substrat montre qu’une protéine recombinante de l’Arabidopsis catalyse la conversion du glyoxylate en en glycolate (Km,glyoxylate = 4,5 µmol·L^–1) et du SSA en γ-hydroxybutyrate (Km, SSA = 0,87 mmol·L^–1), via un mécanisme essentiel irréversible, basé sur le NADPH. Les auteurs ont poussé la caractérisation de l’enzyme dans des études de vélocité initiale, d’inhibition en cul-de-sac et d’inhibition par les produits. Le mécanisme cinétique est déclenché par Bi Bi, et implique la combinaison du NADPH avec l’enzyme, avant le glyoxylate ou le SSA, et le relâchement du NADP⁺ avant le glycolate ou le γ-hydroxybutyrate, respectivement. On peut conclure que l’enzyme fonctionne comme une réductase du glyoxylate (EC1.1.1.79) dépendant du NADPH, ou possiblement une réductase de l’aldéhyde (EC 1.1.1.2), et le mécanisme cinétique impliqué correspond à celui qu’on trouve chez les membres des deux super familles d’enzymes aldo-céto réductases ou reliées à la 3-hydroxyisobutyrate déshydrogénase. Puisque le NADP⁺ est un inhibiteur compétitif efficace relativement au NADPH (Ki = 1–3 µmol·L^–1), on propose que le ratio NADPH/NADP⁺ contrôle l’activité enzymatique in planta. [ABSTRACT FROM AUTHOR]

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