Kinetic and isotopic characterization of l -proline dehydrogenase from mycobacterium tuberculosis

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.