title: Steady-state kinetic mechanism of the proline:ubiquinone oxidoreductase activity of proline utilization A (PutA) from Escherichia coli.
abstract: The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli performs the oxidation of proline to glutamate in two catalytic steps using separate proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains. In the first reaction, the oxidation of proline is coupled to the reduction of ubiquinone (CoQ) by the PRODH domain, which has a β(8)α(8)-barrel structure that is conserved in bacterial and eukaryotic PRODH enzymes. The structural requirements of the benzoquinone moiety were examined by steady-state kinetics using CoQ analogs. PutA displayed activity with all the analogs tested; the highest k(cat)/K(m) was obtained with CoQ(2). The kinetic mechanism of the PRODH reaction was investigated use a variety of steady-state approaches. Initial velocity patterns measured using proline and CoQ(1), combined with dead-end and product inhibition studies, suggested a two-site ping-pong mechanism for PutA. The kinetic parameters for PutA were not strongly influenced by solvent viscosity suggesting that diffusive steps do not significantly limit the overall reaction rate. In summary, the kinetic data reported here, along with analysis of the crystal structure data for the PRODH domain, suggest that the proline:ubiquinone oxidoreductase reaction of PutA occurs via a rapid equilibrium ping-pong mechanism with proline and ubiquinone binding at two distinct sites.
authors: Department of Biochemistry, University of Nebraska-Lincoln, United States.
authors: Michael A
authors: MA
authors: Moxley
authors: John J
authors: JJ
authors: Tanner
authors: Donald F
authors: DF
authors: Becker
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D011392:Proline / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011809:Quinones / Q000378:metabolism / Q000494:pharmacology; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry; D014783:Viscosity
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C080683:PutA protein, Bacteria; D011809:Quinones; D012997:Solvents; D014364:Tryptophan; D011392:Proline