Abstract
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.