Interaction of apo-cytochrome b5 with cytochromes P4503A4 and P45017A:: Relevance of heme transfer reactions

被引:72
作者
Guryev, OL [1 ]
Gilep, AA [1 ]
Usanov, SA [1 ]
Estabrook, RW [1 ]
机构
[1] Univ Texas, SW Med Ctr, Dept Biochem, Dallas, TX 75390 USA
关键词
D O I
10.1021/bi002305w
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 bs 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-bs on the ability of purified CYP3A4 to catalyze the 6 beta -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 GP-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 17 alpha -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-bs, 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.
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页码:5018 / 5031
页数:14
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