title: Kinetic and isotopic characterization of L-proline dehydrogenase from Mycobacterium tuberculosis.
abstract: The monofunctional proline dehydrogenase (ProDH) from Mycobacterium tuberculosis performs the flavin-dependent oxidation of l-proline to Δ(1)-pyrroline-5-carboxylate in the proline catabolic pathway. The ProDH gene, prub, was cloned into the pYUB1062 vector, and the C-terminal His-tagged 37 kDa protein was expressed and purified by nickel affinity chromatography. A steady-state kinetic analysis revealed a ping-pong mechanism with an overall kcat of 33 ± 2 s(-1) and Km values of 5.7 ± 0.8 mM and 3.4 ± 0.3 μM for l-proline and 2,6-dichlorophenolindophenol (DCPIP), respectively. The pH dependence of kcat revealed that one enzyme group exhibiting a pK value of 6.8 must be deprotonated for optimal catalytic activity. Site-directed mutagenesis suggests that this group is Lys110. The primary kinetic isotope effects on V/KPro and V of 5.5 and 1.1, respectively, suggest that the transfer of hydride from l-proline to FAD is rate-limiting for the reductive half-reaction, but that FAD reoxidation is the rate-limiting step in the overall reaction. Solvent and multiple kinetic isotope effects suggest that l-proline oxidation occurs in a stepwise rather than concerted mechanism. Pre-steady-state kinetics reveal an overall kred of 88.5 ± 0.7 s(-1), and this rate is subject to a primary kinetic isotope effect of 5.2. These data confirm that the overall reaction is limited by reduced flavin reoxidation in the second half-reaction.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Hector
authors.initials: H
authors.lastname: Serrano
authors.forename: John S
authors.initials: JS
authors.lastname: Blanchard
mesh_terms: D015086:2,6-Dichloroindophenol / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011394:Proline Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D007554:Isotopes; D015086:2,6-Dichloroindophenol; D011394:Proline Oxidase