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

NAD(P)H:(quinone-acceptor) oxidoreductase (NQOR, EC 1.6.99.2), an enzy me catalyzing the obligatory two electron reduction of quinones, can u tilize both NADH and NADPH as electron donors at similar efficiencies. Based on site-directed mutagenesis studies, we previously suggested t hat the glycine-rich region of rat liver NQOR is important for the bin ding of NAD(P)H (Ma et al., J. Biol. Chem. 267, 22298-22304, 1992). Ho wever, the mode of interactions between the active site and NADH or NA DPH is not clearly known. In this study, we conducted site directed mu tagenesis experiments and identified H194 and D163 of NQOR as key resi dues 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-, an d 96-fold higher, respectively, than that of NQOR; but the K-m(NADH) v alues 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 o f these mutant enzymes were 30 to 60% that of NQOR. In the reduction o f menadione, the mutations also caused much larger increases in k(m(NA DPH)) than K-m(NADH). The results suggest that H194 and D163 are impor tant for the interaction with the 2'-phosphate group of NADPH. NAD(P)H analogues, N-methyldihydronicotinamide and dihydronicotinamide mononu cleotide, can also serve as electron donors for NQOR, but the K-m valu es were 4.5- and 495-fold higher, respectively, than that with NADH. M utations at H194 and D163 and at the glycine rich region of NQOR, whic h 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'-phosphat e group with H194 and D163. (C) 1995 Academic Press, Inc.