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

High Resolution Image Download MS PowerPoint Slide Human 4-oxo- l -proline reductase ( Hs BDH2), 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 Hs BDH2 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 4 S -[ 2 H] accumulating on cis -4-hydroxy- l -proline and the 4 R -[ 2 H] on NADH. Primary kinetic isotope effects, with 4 S -[4- 2 H]NADH, on k cat ( D k cat ) and k cat / K M ( D ( k cat / K M 4OLP )) were small, indicating that hydride transfer is fast relative to other catalytic steps. Internal-competition equilibrium binding isotope effects were inverse with 4 S -[4- 2 H]NADH and normal with 4 R -[4- 2 H]NADH, suggesting that Hs BDH2 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 ( k cat / K M ) of 2.6 and a D k cat of 2.5 were determined with the former, modest values were obtained with the latter. Using NADPH as a coenzyme resulted in a similar k cat, but drastically increased K M . Accordingly, 4 S -[4- 2 H]NADPH increased D ( k cat / K M NADPH ) to 1.5, while D k cat remained small. The N105A substitution, proposed to affect coenzyme binding in SDRs, increased K M for NADH and doubled k cat . The D ( k cat / K M ) increased to 2.5 and D k cat to 2.0. The D 2 O k cat with 4 S -[4- 2 H]NADH decreased from its value with NADH, suggesting stepwise hydride-transfer and proton-transfer steps. These results expand the role of N105 in Hs BDH2 and possibly other SDRs.