ROLES OF HISTIDINE-194, ASPARTATE-163, AND A GLYCINE-RICH SEQUENCE OF NAD(P)H-QUINONE OXIDOREDUCTASE IN THE INTERACTION WITH NICOTINAMIDE COENZYMES

NAD(P)H:(quinone-acceptor) oxidoreductase (NQOR, EC 1.6.99.2), an enzyme catalyzing the obligatory two electron reduction of quinones, can utilize both NADH and NADPH as electron donors at similar efficiencies. Based on site-directed mutagenesis studies, we previously suggested that the glycine-rich region of rat liver NQOR is important for the binding of NAD(P)H (Ma et al., J. Biol. Chem. 267, 22298-22304, 1992). However, the mode of interactions between the active site and NADH or NADPH is not clearly known. In this study, we conducted site directed mutagenesis experiments and identified H194 and D163 of NQOR as key residues affecting the K-m of NADPH. Steady-state kinetic analysis for the reduction of dichloroindophenol (DCIP) showed that K-m(NADPH) values of purified mutant proteins H194D, H194A, and D163V were 288-, 14-, and 96-fold higher, respectively, than that of NQOR; but the K-m(NADH) values were only slightly higher. The k(cat(NADPH)) values were almost the same as that of NQOR in the reduction of DCIP at the respective pH optima which were affected by the mutations. The K-cat(NADH) values of these mutant enzymes were 30 to 60% that of NQOR. In the reduction of menadione, the mutations also caused much larger increases in k(m(NADPH)) than K-m(NADH). The results suggest that H194 and D163 are important for the interaction with the 2'-phosphate group of NADPH. NAD(P)H analogues, N-methyldihydronicotinamide and dihydronicotinamide mononucleotide, can also serve as electron donors for NQOR, but the K-m values were 4.5- and 495-fold higher, respectively, than that with NADH. Mutations at H194 and D163 and at the glycine rich region of NQOR, which increased K-m(NADH) and K-m(NADPH), did not substantially affect the K-m values of these two analogues. This result is consistent with the suggested roles of these amino acid residues in the interaction with nicotinamide coenzymes. Based on these results, a model of the NAD(P)H binding site is proposed showing the interaction of the pyrophosphate group with the glycine-rich region and the interaction of 2'-phosphate group with H194 and D163. (C) 1995 Academic Press, Inc.