Kinetic and Thermodynamic Characterization of Human 4-Oxo-l-proline Reductase Catalysis.

The enzyme 4-oxo-l-proline reductase (BDH2) has recently been identified in humans. BDH2, previously thought to be a cytosolic (R)-3-hydroxybutyrate dehydrogenase, actually catalyzes the NADH-dependent reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline, a compound with known anticancer activity. Here we provide an initial mechanistic characterization of the BDH2-catalyzed reaction. Haldane relationships show the reaction equilibrium strongly favors the formation of cis-4-hydroxy-l-proline. Stereospecific deuteration of NADH C4 coupled with mass spectrometry analysis of the reaction established that the pro-S hydrogen is transferred. NADH is co-purified with the enzyme, and a binding kinetics competition assays with NAD⁺ defined dissociation rate constants for NADH of 0.13 s^-1 at 5 °C and 7.2 s^-1 at 25 °C. Isothermal titration calorimetry at 25 °C defined equilibrium dissociation constants of 0.48 and 29 μM for the BDH2:NADH and BDH2:NAD⁺ complexes, respectively. Differential scanning fluorimetry showed BDH2 is highly thermostabilized by NADH and NAD⁺. The k_cat/K_M pH-rate profile indicates that a group with a pK_a of 7.3 and possibly another with a pK_a of 8.7 must be deprotonated and protonated, respectively, for maximum binding of 4-oxo-l-proline and/or catalysis, while the k_cat profile is largely insensitive to pH in the pH range used. The single-turnover rate constant is only 2-fold higher than k_cat. This agrees with a pre-steady-state burst of substrate consumption, suggesting that a step after chemistry, possibly product release, contributes to limit k_cat. A modest solvent viscosity effect on k_cat indicates that this step is only partially diffusional. Taken together, these data suggest chemistry does not limit the reaction rate but may contribute to it.