Electrostatic interaction between cytochrome P450 and NADPH-P450 reductase: comparison of mixed and fused systems consisting of rat cytochrome P450 1A1 and yeast NADPH-P450 reductase.
The electrostatic interaction between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase was analyzed by using recombinant yeast microsomes containing both native enzymes or their fused enzyme. The Vmax of the 7-ethoxycoumarin O-deethylation in the recombinant microsomes containing both rat cytochrome P4501A1 and yeast NADPH-P450 reductase (the mixed system) was maximal when the ionic strength of the reaction mixture was 0.1-0.15. However, on the fused enzyme between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase (the fused system), the activity was uniformly reduced with increasing ionic strength. The pH profiles of Vmax were also different between the mixed and the fused systems. Based on these results, we propose a hypothesis that cytochrome P450 and NADPH-P450 reductase have more than one binding mode. The maximal activity of the mixed system at ionic strength of 0.1-0.15 is explained by change of the binding mode. On the other hand, the fused enzyme appears to have only one binding mode due to the limited topology of cytochrome P450 and NADPH-P450 reductase domains.
Kondo
S
S
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan.
Sakaki
T
T
Ohkawa
H
H
Inouye
K
K
D000818:Animals; D002384:Catalysis / Q000187:drug effects; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D009994:Osmolar Concentration; D010084:Oxidation-Reduction / Q000187:drug effects; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012965:Sodium Chloride / Q000494:pharmacology; D055672:Static Electricity
D003374:Coumarins; D011993:Recombinant Fusion Proteins; C017299:7-ethoxycoumarin; D012965:Sodium Chloride; D011189:Potassium Chloride; D003577:Cytochrome P-450 Enzyme System; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase