Strittmatter P, Kittler J M, Coghill J E, Ozols J
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030.
J Biol Chem. 1992 Feb 5;267(4):2519-23.
An expression vector for bovine NADH-cytochrome b5 reductase was constructed from two DNA fragments that were derived from beef liver poly(A+) RNA using the polymerase chain reaction. Site-directed mutagenesis of the 3 lysine residues of the reductase, previously implicated in the formation of active-site charge pairs with carboxylate residues of cytochrome b5, was then used to obtain the purified catalytic domains of flavoproteins modified at each of these sites. The observed marked decreases in catalytic efficiencies of substitutions of a negative charge at the normally positively charged residues with the catalytic domain of cytochrome b5 are consistent with their participation in the formation of charge pairs with carboxylate groups of the hemeprotein to optimize rapid electron transfer from the reductase flavin to the heme of the cytochrome.
利用聚合酶链反应从牛肉肝聚腺苷酸（poly(A+)）RNA衍生的两个DNA片段构建了牛NADH-细胞色素b5还原酶的表达载体。还原酶的3个赖氨酸残基先前被认为与细胞色素b5的羧酸盐残基形成活性位点电荷对，然后通过定点诱变获得在这些位点各自修饰的黄素蛋白的纯化催化结构域。观察到在正常带正电荷的残基处用细胞色素b5的催化结构域取代负电荷后催化效率显著降低，这与它们参与与血红素蛋白的羧酸盐基团形成电荷对以优化从还原酶黄素到细胞色素血红素的快速电子转移是一致的。
