title: Cytochrome b5 Activates the 17,20-Lyase Activity of Human Cytochrome P450 17A1 by Increasing the Coupling of NADPH Consumption to Androgen Production.
abstract: Human cytochrome P450 17A1 is required for all androgen biosynthesis and is the target of abiraterone, a drug used widely to treat advanced prostate cancer. P450 17A1 catalyzes both 17-hydroxylation and subsequent 17,20-lyase reactions with pregnenolone, progesterone, and allopregnanolone. The presence of cytochrome b5 (b5) markedly stimulates the 17,20-lyase reaction, with little effect on 17-hydroxylation; however, the mechanism of this b5 effect is not known. We determined the influence of b5 on coupling efficiency-defined as the ratio of product formation to NADPH consumption-in a reconstituted system using these 3 pairs of substrates for the 2 reactions. Rates of NADPH consumption ranged from 4 to 13 nmol/min/nmol P450 with wild-type P450 17A1. For the 17-hydroxylase reaction, progesterone oxidation was the most tightly coupled (∼50%) and negligibly changed upon addition of b5. Rates of NADPH consumption were similar for the 17-hydroxylase and corresponding 17,20-lyase reactions for each steroid series, and b5 only slightly increased NADPH consumption. For the 17,20-lyase reactions, b5 markedly increased product formation and coupling in parallel with all substrates, from 6% to 44% with the major substrate 17-hydroxypregnenolone. For the naturally occurring P450 17A1 mutations E305G and R347H, which impair 17,20-lyase activity, b5 failed to rescue the poor coupling with 17-hydroxypregnenolone (2-4%). When the conserved active-site threonine was mutated to alanine (T306A), both the activity and coupling were markedly decreased with all substrates. We conclude that b5 stimulation of the 17,20-lyase reaction primarily derives from more efficient use of NADPH for product formation rather than side products.
authors.forename: Hwei-Ming
authors.initials: HM
authors.lastname: Peng
authors.affiliation: Department of Anesthesiology, University of Michigan and the VA Medical Center , 2215 Fuller Road, Building 31, Room 225, Ann Arbor, Michigan 48105, United States.
authors.forename: Sang-Choul
authors.initials: SC
authors.lastname: Im
authors.affiliation: Department of Anesthesiology, University of Michigan and the VA Medical Center , 2215 Fuller Road, Building 31, Room 225, Ann Arbor, Michigan 48105, United States.
authors.forename: Naw May
authors.initials: NM
authors.lastname: Pearl
authors.forename: Adina F
authors.initials: AF
authors.lastname: Turcu
authors.forename: Juilee
authors.initials: J
authors.lastname: Rege
authors.affiliation: Department of Anesthesiology, University of Michigan and the VA Medical Center , 2215 Fuller Road, Building 31, Room 225, Ann Arbor, Michigan 48105, United States.
authors.forename: Lucy
authors.initials: L
authors.lastname: Waskell
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Auchus
mesh_terms: D019943:Amino Acid Substitution; D000728:Androgens / Q000096:biosynthesis*; D000736:Androstenes / Q000494:pharmacology; D020134:Catalytic Domain; D015786:Cytochromes b5 / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000728:Androgens; D000736:Androstenes; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D011994:Recombinant Proteins; D009249:NADP; D015786:Cytochromes b5; C501892:CYP17A1 protein, human; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; C089740:abiraterone