Effects of ionic strength on the functional interactions between CYP2B4 and CYP1A2.
The presence of one P450 can influence the catalytic characteristics of a second enzyme through the formation of heteromeric P450 complexes. Such a complex has been reported for mixed reconstituted systems containing NADPH-cytochrome P450 reductase, CYP2B4, and CYP1A2, where a dramatic inhibition of 7-pentoxyresorufin-O-dealkylation (PROD) was observed when compared to simple reconstituted systems containing reductase and a single P450 enzyme. The goal of the present study was to characterize this interaction by examining the potential of the CYP1A2-CYP2B4 complex to be formed by charge-pair interactions. With ionic interactions being sensitive to the surrounding ionic environment, monooxygenase activities were measured in both simple systems and mixed reconstituted systems as a function of ionic strength. PROD was found to be decreased at high ionic strength in both simple and mixed reconstituted systems, due to disruption of reductase-P450 complexes. Additionally, the inhibition of PROD in mixed reconstituted systems was relieved at high ionic strength, consistent with disruption of the CYP2B4-CYP1A2 complex. When ionic strength was measured as a function of CYP1A2 concentration, a shift to the right in the inflection point of the biphasic curve occurred at high ionic strength, consistent with a loss in CYP1A2 affinity for CYP2B4. When this analysis was applied to the same systems using a different substrate, 7-EFC, evidence for a high-affinity complex was not observed, demonstrating that the characteristics of the CYP1A2-CYP2B4 complex are influenced by the substrates present. These results support the role for a substrate specific electrostatic interaction between these P450 enzymes.
Kelley
Rusty W
RW
Department of Pharmacology and Experimental Therapeutics and The Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, Louisiana 70112, USA.
Reed
James R
JR
Backes
Wayne L
WL
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry* / Q000378:metabolism*; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D000072467:Cytochrome P450 Family 2; D003640:Dealkylation; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009994:Osmolar Concentration; D010078:Oxazines / Q000378:metabolism; D011817:Rabbits; D055672:Static Electricity; D013379:Substrate Specificity
D003374:Coumarins; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D009097:Multienzyme Complexes; D010078:Oxazines; C056033:7-ethoxy-4-trifluoromethylcoumarin; C044943:pentoxyresorufin; D001189:Aryl Hydrocarbon Hydroxylases; D019388:Cytochrome P-450 CYP1A2; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D009251:NADPH-Ferrihemoprotein Reductase; D008274:Magnesium