title: Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1).
abstract: Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.
authors.affiliation: School of Chemistry, Monash University, Clayton, Victoria, Australia.
authors.forename: Alexandr N
authors.initials: AN
authors.lastname: Simonov
authors.affiliation: ACRF Rational Drug Discovery Centre, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
authors.forename: Jessica K
authors.initials: JK
authors.lastname: Holien
authors.affiliation: School of Chemistry, Monash University, Clayton, Victoria, Australia.
authors.forename: Joyee Chun In
authors.initials: JC
authors.lastname: Yeung
authors.affiliation: School of Veterinary Medicine, University of California Davis, Davis, California, United States of America.
authors.forename: Ann D
authors.initials: AD
authors.lastname: Nguyen
authors.affiliation: School of Veterinary Medicine, University of California Davis, Davis, California, United States of America.
authors.forename: C Jo
authors.initials: CJ
authors.lastname: Corbin
authors.affiliation: Department of Physiology and Membrane Biology, School of Medicine, University of California Davis, Davis, California, United States of America.
authors.forename: Jie
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Boreskov Institute of Catalysis, Prospekt Lavrentieva 5, Novosibirsk, Russia.
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Kuznetsov
authors.affiliation: Division of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Auchus
authors.affiliation: School of Veterinary Medicine, University of California Davis, Davis, California, United States of America.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Conley
authors.affiliation: School of Chemistry, Monash University, Clayton, Victoria, Australia.
authors.forename: Alan M
authors.initials: AM
authors.lastname: Bond
authors.affiliation: ACRF Rational Drug Discovery Centre, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.|Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Parker
authors.affiliation: Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive Health, Robinson Research Institute, University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Rodgers
authors.affiliation: School of Chemistry, Monash University, Clayton, Victoria, Australia.
authors.forename: Lisandra L
authors.initials: LL
authors.lastname: Martin
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D009154:Mutation; D037742:Nanotubes, Carbon / Q000737:chemistry; D011485:Protein Binding; D060066:Protein Interaction Maps / Q000235:genetics*; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D037742:Nanotubes, Carbon; D015786:Cytochromes b5; C501892:CYP17A1 protein, human; D013254:Steroid 17-alpha-Hydroxylase