Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization a protein

Michael A. Moxley, Donald F Becker

Research output: Contribution to journalArticle

19 Citations (Scopus)

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 Δ 1- 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 CoQ 1 (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 CoQ 1, 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 k cat during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental k cat and k cat/K m parameters.

Original languageEnglish (US)
Pages (from-to)511-520
Number of pages10
JournalBiochemistry
Volume51
Issue number1
DOIs
StatePublished - Jan 10 2012

Fingerprint

Proline Oxidase
Proline
Reaction kinetics
Rate constants
Isomerization
Proteins
Cats
Ubiquinone
1-Pyrroline-5-Carboxylate Dehydrogenase
Oxidation
Kinetics
Biochemistry
Escherichia coli
Glutamic Acid
Oxidoreductases
4,6-dinitro-o-cresol

ASJC Scopus subject areas

  • Biochemistry

Cite this

Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization a protein. / Moxley, Michael A.; Becker, Donald F.

In: Biochemistry, Vol. 51, No. 1, 10.01.2012, p. 511-520.

Research output: Contribution to journalArticle

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