Interaction of apo-cytochrome b5 with cytochromes P4503A4 and P45017A: relevance of heme transfer reactions.
Maximal activity of CYP3A4 is obtained using a reconstitution system consisting of NADPH-P450 reductase (CPR), dioleoylphosphatidylcholine (DOPC), an ionic detergent, and cytochrome b(5) (b(5)). The mechanism by which b(5) stimulates the catalytic activity of CYP3A4 is controversial. Recent data report that apo-cytochrome b(5) (apo-b(5)) can substitute for holo-b(5) by serving as an allosteric effector. These authors concluded that b(5) is not directly involved in electron transfer reactions to CYP3A4. We have studied the effect of apo-b(5) on the ability of purified CYP3A4 to catalyze the 6beta-hydroxylation of testosterone and horse CYP17A to catalyze the 17,20-lyase reaction. The high molecular weight form of holo-b(5) (HMW-holo-b(5)) stimulates the 6beta-hydroxylation of testosterone while the low molecular weight (truncated) form of holo-b(5) (LMW-holo-b(5)) does not. When added to the reconstituted system, HMW-apo-b(5) stimulates the activity of CYP3A4 to a level 50-60% of that obtained with HMW-holo-b(5). A similar stimulation of 17alpha-hydroxyprogesterone metabolism is seen when studying the CYP17A-catalyzed reaction. Neither LMW-holo-b(5) nor LMW-apo-b(5) stimulates the activity of CYP3A4 or CYP17A. CYP3A4 forms a complex during affinity chromatography with immobilized HMW-holo-b(5) but not with immobilized HMW-apo-b(5). Incubation of apo-b(5) with CYP3A4, using conditions required for reconstitution of enzymatic activities, results in the transfer of heme from the CYP3A4 preparation to apo-b(5), thereby forming holo-b(5). The separation of heme proteins by thiol-disulfide exchange chromatography confirms the formation of holo-b(5). A His67Ala mutant of HMW-b(5) as well as the Zn-substituted protoporphyrin derivative of HMW-b(5) do not stimulate the activity of either CYP3A4 or CYP17A. These data show that the mechanism of stimulation of CYP3A4 and CYP17A activities by apo-b(5) results from the formation of holo-b(5) by a heme transfer reaction.
Guryev
O L
OL
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA.
Gilep
A A
AA
Usanov
S A
SA
Estabrook
R W
RW
D000409:Alanine / Q000235:genetics; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases; D002384:Catalysis; D002846:Chromatography, Affinity; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism; D045303:Cytochromes b; D004579:Electron Transport / Q000235:genetics; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D006801:Humans; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry* / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000737:chemistry
D001059:Apoproteins; D003573:Cytochrome b Group; D004800:Enzymes, Immobilized; D020035:Holoenzymes; D009211:Myoglobin; D011994:Recombinant Proteins; C010857:apomyoglobin; D006418:Heme; D006639:Histidine; D045303:Cytochromes b; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C022724:testosterone 7-alpha-hydroxylase, hamster; D013254:Steroid 17-alpha-Hydroxylase; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase; D000409:Alanine