Kinetic and Isotopic Characterization of l‑Proline Dehydrogenase from Mycobacterium tuberculosis

• Published in: Biochemistry (Easton) Vol. 52; no. 29; pp. 5009 - 5015
• Main Authors: Serrano, Hector, Blanchard, John S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 23.07.2013
• Subjects: 2,6-Dichloroindophenol - chemistry; affinity chromatography; Amino Acid Sequence; Base Sequence; catalytic activity; Cloning, Molecular; DNA Primers; genes; hydrides; Hydrogen-Ion Concentration; Isotopes; Kinetics; Molecular Sequence Data; Mutation; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; nickel; oxidation; Polymerase Chain Reaction; proline; Proline Oxidase - chemistry; Proline Oxidase - genetics; Proline Oxidase - metabolism; Sequence Homology, Amino Acid; site-directed mutagenesis; solvents
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/bi400338f

The monofunctional proline dehydrogenase (ProDH) from Mycobacterium tuberculosis performs the flavin-dependent oxidation of l-proline to Δ1-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 k cat of 33 ± 2 s–1 and K m 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 k cat 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/K Pro 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 k red 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.