INTERACTION WITH ARGININE-597 OF NADPH CYTOCHROME-P-450 OXIDOREDUCTASE IS A PRIMARY SOURCE OF THE UNIFORM BINDING-ENERGY USED TO DISCRIMINATE BETWEEN NADPH AND NADH - APPENDIX - THERMODYNAMIC INTERPRETATION OFCOMBINED ALTERNATE SUBSTRATE INHIBITOR, PH, AND SITE-DIRECTED MUTAGENESIS STUDIES

Site-directed mutagenesis has been used in conjunction with pH and alt ernate substrate/inhibitor studies to characterize the interactions be tween NADPH-cytochrome P-450 oxidoreductase (P-450R) and the 2'-phosph ate of NADP(H) that provide P-450R with its strong nicotinamide nucleo tide specificity. It is known that the 2'-phosphate of NADP(H) is boun d to P-450R as the dianion and that interactions between it and residu es 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 obser vation of similar P-31-NMR chemical shifts for FAD (pyrophosphate) bou nd to it and wild-type (WT) P-450R, although it is more unstable to ur ea denaturation. P-450R (R597M) has a K(m) for NADPH that is 150 times that of P-450R (WT) and a K(i) for NADP+ that is 240 times that of P- 450R (WT). In contrast, the R597M mutation has only a modest effect on the K(m) for NADH (0.8WT) and the K(i) for NAD+ (2.9WT), indicating t hat Arg597 must have been interacting specifically with the 2'-phospha te of NADP(H). The R597M mutation has relatively little effect on k(ca t) for NADPH (1.2WT) or NADH (0.6WT), 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+ pK(i) pro file for P-450R (R597M) shows a pK(a) of 5.78 for the 2'-phosphate of NADP+, which is bound to P-450R (R597M) as the dianion, but the pK(a) 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 pK(a) of 9.5. Finally, a general theoretical treatmen t of the thermodynamic consequences of individual and combined perturb ations to complementary interacting groups on enzyme and substrate is presented (see Appendix). This allows conclusions to be made regarding the severity of the structural perturbations resulting from a mutatio n: local (affecting interactions with one group on the substrate), pro ximal (affecting interactions with the rest of the substrate), or glob al (affecting interactions in another binding site).