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• W2019664503 endingPage "520" @default.
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• W2019664503 abstract "The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ1 (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s–1 was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s–1. The isomerization step is proposed to report on a previously identified flavin-dependent conformational change [Zhang, W. et al. (2007) Biochemistry 46, 483–491] that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ1, a soluble analogue of ubiquinone, a rate constant of 5.4 s–1 was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for kcat during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental kcat and kcat/Km parameters." @default.
• W2019664503 created "2016-06-24" @default.
• W2019664503 creator A5005735997 @default.
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• W2019664503 date "2011-12-15" @default.
• W2019664503 modified "2023-10-18" @default.
• W2019664503 title "Rapid Reaction Kinetics of Proline Dehydrogenase in the Multifunctional Proline Utilization A Protein" @default.
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• W2019664503 doi "https://doi.org/10.1021/bi201603f" @default.
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