Journal
BIOCHEMISTRY
Volume 40, Issue 16, Pages 5018-5031Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi002305w
Keywords
-
Categories
Funding
- NIGMS NIH HHS [GM 16488-32] Funding Source: Medline
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available