Ma Q, Cui K, Xiao F, Lu A Y, Yang C S
Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855.
J Biol Chem. 1992 Nov 5;267(31):22298-304.
Site-directed mutagenesis was utilized to identify binding sites for NAD(P)H and dicumarol in rat liver NAD(P)H:quinone oxidoreductase (NQOR, EC 1.6.99.2). The mutant cDNA clones were generated by a procedure based on the polymerase chain reaction and were expressed in Escherichia coli. The mutant enzymes were purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and were found to contain 2 FADs/enzyme molecule identical with that of the wild-type NQOR. Purified mutant enzymes Y128D, G150F, G150V, S151F, and Y155D showed dramatic decreases in activities in the reduction of dichlorophenolindophenol in comparison with the activities of the wild-type enzyme, whereas the activities of F124L, T127V, T127E, Y128V, Y128F, S151A, and Y155V were similar to those of NQOR. Enzyme kinetic analysis revealed that the Km values of T127E, Y128D, G150F, G150V, S151F, and Y155D were, respectively, 4-, 2-, 13-, 5-, 26-, and 19-fold higher than the Km of NQOR for NADPH, and were, respectively, 2-, 3-, 7-, 3-, 20-, and 11-fold higher than that of NQOR for NADH. The kcat values of Y128D, G150F, and G150V were also much lower than those of NQOR, but the kcat values of other mutants were similar to those of the wild-type enzyme. The Km values of the mutants for dichlorophenolindophenol were the same or slightly higher than that of NQOR. The apparent inhibition constants (Ki) for dicumarol on Y128V and F124L were elevated 12 and 8 times, respectively. Similar, but smaller, changes on Ki for 4-hydroxycoumarin were also observed. This study demonstrated that residues Gly150, Ser151, and Tyr155 in the glycine-rich region of NQOR are essential for NADPH and NADH binding and Tyr128 is important for dicumarol binding. Based on the results of the study, it is proposed that the glycine-rich region of the enzyme, along with other residues around the region, forms a beta sheet-turn-alpha helix structure important for the binding of the pyrophosphate group of NADPH and NADH.
利用定点诱变技术确定大鼠肝脏NAD(P)H:醌氧化还原酶（NQOR，EC 1.6.99.2）中NAD(P)H和双香豆素的结合位点。通过基于聚合酶链反应的方法生成突变体cDNA克隆，并在大肠杆菌中表达。经SDS-聚丙烯酰胺凝胶电泳判断，突变酶被纯化至表观均一，且发现每个酶分子含有2个FAD，与野生型NQOR相同。与野生型酶的活性相比，纯化的突变酶Y128D、G150F、G150V、S151F和Y155D在还原二氯酚靛酚时活性显著降低，而F124L、T127V、T127E、Y128V、Y128F、S151A和Y155V的活性与NQOR相似。酶动力学分析表明，T127E、Y128D、G150F、G150V、S151F和Y155D对NADPH的Km值分别比NQOR高4、2、13、5、26和19倍，对NADH的Km值分别比NQOR高2、3、7、3、20和11倍。Y128D、G150F和G150V的kcat值也远低于NQOR，但其他突变体的kcat值与野生型酶相似。突变体对二氯酚靛酚的Km值与NQOR相同或略高。双香豆素对Y128V和F124L的表观抑制常数（Ki）分别升高了12倍和8倍。对于4-羟基香豆素的Ki也观察到类似但较小的变化。本研究表明，NQOR富含甘氨酸区域的Gly150、Ser151和Tyr155残基对于NADPH和NADH结合至关重要，而Tyr128对于双香豆素结合很重要。基于该研究结果，提出该酶的富含甘氨酸区域以及该区域周围的其他残基形成了一个β折叠-转角-α螺旋结构，对NADPH和NADH焦磷酸基团的结合很重要。
