Sem D S, Kasper C B
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Biochemistry. 1993 Nov 2;32(43):11548-58. doi: 10.1021/bi00094a011.
Site-directed mutagenesis has been used in conjunction with pH and alternate substrate/inhibitor studies to characterize the interactions between NADPH-cytochrome P-450 oxidoreductase (P-450R) and the 2'-phosphate of NADP(H) that provide P-450R with its strong nicotinamide nucleotide specificity. It is known that the 2'-phosphate of NADP(H) is bound to P-450R as the dianion and that interactions between it and residues on P-450R provide 5 kcal/mol of essentially uniform binding energy (preceding paper in this issue). In order to probe these interactions further, Arg597 of P-450R, which is homologous to Arg235 of ferredoxin-NADP+ reductase that forms a salt bridge with the 2'-phosphate of 2'-phospho-AMP in the crystal structure of that complex [Karplus, P. A., Daniels, M. J., & Herriott, J. R. (1991) Science 251, 60], was mutated to methionine. The mutant protein, P-450R (R597M), does not appear to have a grossly perturbed tertiary structure on the basis of the observation of similar 31P-NMR chemical shifts for FAD (pyrophosphate) bound to it and wild-type (WT) P-450R, although it is more unstable to urea denaturation. P-450R (R597M) has a Km for NADPH that is 150 times that of P-450R (WT) and a Ki for NADP+ that is 240 times that of P-450R (WT). In contrast, the R597M mutation has only a modest effect on the Km for NADH (0.8 WT) and the Ki for NAD+ (2.9 WT), indicating that Arg597 must have been interacting specifically with the 2'-phosphate of NADP(H). The R597M mutation has relatively little effect on kcat for NADPH (1.2 WT) or NADH (0.6 WT), indicating that the mutation is affecting ground and transition states to essentially the same degree, by removing 3 kcal/mol of uniform binding energy. The NADP+ pKi profile for P-450R (R597M) shows a pKa of 5.78 for the 2'-phosphate of NADP+, which is bound to P-450R (R597M) as the dianion, but the pKa of 9.5 for the preferentially protonated enzymic group observed in the P-450R (WT) profile is no longer present. It is argued then that the 2'-phosphate binding pocket of P-450R (WT) has a high positive charge density (> + 2) and that Arg597, which is in this binding pocket, has a highly perturbed pKa of 9.5. Finally, a general theoretical treatment of the thermodynamic consequences of individual and combined perturbations to complementary interacting groups on enzyme and substrate is presented (see Appendix).(ABSTRACT TRUNCATED AT 400 WORDS)
定点诱变已与pH值及替代底物/抑制剂研究结合使用，以表征NADPH - 细胞色素P - 450氧化还原酶（P - 450R）与NADP(H)的2'-磷酸之间的相互作用，正是这些相互作用赋予了P - 450R强烈的烟酰胺核苷酸特异性。已知NADP(H)的2'-磷酸以二价阴离子形式与P - 450R结合，并且它与P - 450R上的残基之间的相互作用提供了5千卡/摩尔基本均匀的结合能（本期前文）。为了进一步探究这些相互作用，将P - 450R的Arg597突变为甲硫氨酸，该残基与铁氧化还原蛋白 - NADP⁺还原酶的Arg235同源，在该复合物的晶体结构中，后者与2'-磷酸 - AMP的2'-磷酸形成盐桥[卡尔普斯，P. A.，丹尼尔斯，M. J.，& 赫里奥特，J. R.（1991年）《科学》251，60]。基于与结合在其上的FAD（焦磷酸）和野生型（WT）P - 450R类似的³¹P - NMR化学位移观察，突变蛋白P - 450R（R597M）似乎没有明显扰乱的三级结构，尽管它对尿素变性更不稳定。P - 450R（R597M）对NADPH的Km是P - 450R（WT）的150倍，对NADP⁺的Ki是P - 450R（WT）的240倍。相比之下，R597M突变对NADH的Km（0.8倍WT）和NAD⁺的Ki（2.9倍WT）只有适度影响，表明Arg597一定是与NADP(H)的2'-磷酸特异性相互作用。R597M突变对NADPH（1.2倍WT）或NADH（0.6倍WT）的kcat影响相对较小，表明该突变通过去除3千卡/摩尔均匀结合能，对基态和过渡态的影响基本相同。P - 450R（R597M）的NADP⁺ pKi曲线显示，作为二价阴离子与P - 450R（R597M）结合的NADP⁺的2'-磷酸的pKa为5.78，但在P - 450R（WT）曲线中观察到的优先质子化酶基团的9.5的pKa不再存在。由此认为，P - 450R（WT）的2'-磷酸结合口袋具有高正电荷密度（> +2），位于该结合口袋中的Arg597具有高度扰动的9.5的pKa。最后，给出了对酶和底物上互补相互作用基团的单个和组合扰动的热力学后果的一般理论处理（见附录）。（摘要截短于400字）
