Enzyme-substrate binding interactions of NADPH-cytochrome P-450 oxidoreductase characterized with pH and alternate substrate/inhibitor studies.
The pH dependence of the kinetic parameters for the reaction catalyzed by NADPH-cytochrome P-450 oxidoreductase (P-450R) has been determined, using various substrates and inhibitors. All Vmax and (V/K) profiles show pKas of 6.2-7.3, for an acidic group that is preferentially unprotonated for catalysis, and of 8.1-9.6, for a basic group that is preferentially protonated for catalysis. The presence of the wrong ionization state for both of these groups is tolerated more at lower ionic strength (300 mM) than at higher ionic strength (850 mM). Ionization of the basic group has a more pronounced effect on binding of substrate (cytochrome c or dichloroindophenol) than on catalysis, since ionization has only a 2-fold effect on Vmax with cytochrome c, and only a 5-fold effect on Vmax with dichloroindophenol, while (V/K) for both substrates continues to drop at high pH with no sign of reaching a plateau. Therefore, this basic group affects predominantly substrate binding and, to a lesser extent, catalysis. It is most likely located on the surface of the protein at the cytochrome c/dichloroindophenol binding site, near the FMN prosthetic group. The NADP+ pKi profile shows a pKa of 5.95 for the 2'-phosphate of NADP+, which is bound to P-450R as the dianion, and a pKa of 9.53 for an enzyme group that must be protonated in order to bind NADP+.(ABSTRACT TRUNCATED AT 250 WORDS)
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McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Kasper
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D000818:Animals; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009994:Osmolar Concentration; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D009251:NADPH-Ferrihemoprotein Reductase