Probing the Human 4-Oxo-l-proline Reductase-Catalyzed Reaction by Deuterium Kinetic and Equilibrium Isotope Effects

Human 4-oxo-l-proline reductase (HsBDH2), a short-chain dehydrogenase/reductase (SDR) superfamily member possessing a canonical N-S-Y-K catalytic tetrad, catalyzes the formation of the endogenous anticancer compound cis-4-hydroxy-l-proline via the NADH-dependent reduction of 4-oxo-l-proline. Except for hydride-transfer stereochemistry, information on the HsBDH2 chemical step remains elusive. Here, deuterium isotope effects are employed to gather information on the hydride-transfer step of the reaction. Primary and α-secondary equilibrium isotope effects were inverse and normal, respectively, consistent with the 4S-[2H] accumulating on cis-4-hydroxy-l-proline and the 4R-[2H] on NADH. Primary kinetic isotope effects, with 4S-[4-2H]NADH, on kcat (Dkcat) and kcat/KM (D(kcat/KM4OLP)) were small, indicating that hydride transfer is fast relative to other catalytic steps. Internal-competition equilibrium binding isotope effects were inverse with 4S-[4-2H]NADH and normal with 4R-[4-2H]NADH, suggesting that HsBDH2 tight binding distorts the pro-S hydrogen to a more constrained bonding environment, while the opposite happens to the pro-R hydrogen. Both levulinate and pyruvate were low-affinity, slow-reaction substrates; while a D(kcat/KM) of 2.6 and a Dkcat of 2.5 were determined with the former, modest values were obtained with the latter. Using NADPH as a coenzyme resulted in a similar kcat, but drastically increased KM. Accordingly, 4S-[4-2H]NADPH increased D(kcat/KMNADPH) to 1.5, while Dkcat remained small. The N105A substitution, proposed to affect coenzyme binding in SDRs, increased KM for NADH and doubled kcat. The D(kcat/KM) increased to 2.5 and Dkcat to 2.0. The D2Okcat with 4S-[4-2H]NADH decreased from its value with NADH, suggesting stepwise hydride-transfer and proton-transfer steps. These results expand the role of N105 in HsBDH2 and possibly other SDRs.