title: Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization A protein.
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.
authors.affiliation: Department of Biochemistry and Redox Biology Center, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, United States.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Moxley
authors.forename: Donald F
authors.initials: DF
authors.lastname: Becker
mesh_terms: D050842:1-Pyrroline-5-Carboxylate Dehydrogenase / Q000737:chemistry / Q000502:physiology; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D020369:Bradyrhizobium / Q000201:enzymology; D002384:Catalysis; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006706:Homeostasis / Q000502:physiology; D007536:Isomerism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology; D010084:Oxidation-Reduction; D011392:Proline / Q000737:chemistry*; D011394:Proline Oxidase / Q000737:chemistry* / Q000502:physiology; D055503:Protein Multimerization / Q000502:physiology; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C080683:PutA protein, Bacteria; D005182:Flavin-Adenine Dinucleotide; D018698:Glutamic Acid; D011392:Proline; D050842:1-Pyrroline-5-Carboxylate Dehydrogenase; D011394:Proline Oxidase