PARTICIPATION IN CYTOCHROME B5 IN ANESTHETIC METABOLISM
Lucy A Waskell
University Of Michigan At Ann Arbor, 1040 Wolverine Tower, Ann Arbor, Mi 48109-1274
Grant 5R01GM035533-22 from National Institute Of General Medical Sciences
Abstract: The cytochromes P450 (cyts P450) are a ubiquitous superfamily of oxidases whose activity is influenced by a second protein, cytochrome b5 (cyt b5). The long-term goal of this project is to understand the structural and mechanistic basis of the modification of human cyt P450-mediated metabolism by cyt b5 in order to develop and employ strategies to modify the activity of these essential isozymes for human benefit. In order to understand the molecular mechanism by which cyt b5 influences oxidation by cyt P450 in vivo, one must understand the details of how cyt b5 interacts with cyt P450 and how it modifies the catalytic cycle of cyt P450 relative to cyt P450 reductase. There are more than 8,000 members of the cyts P450 superfamily. They are found in all kingdoms of living organisms and plants and are referred to as Mother Nature´s blowtorch, due to their ability to oxidize a vast number of stable chemical entities. Humans possess 56 different cyts P450, many of which are essential for early development and life itself. Other human cyts P450 determine the toxicity, duration of action, and elimination of the vast majority of therapeutic agents, carcinogens, and environmental agents to which humans are exposed. Xenobiotic metabolizing cyts P450 are also responsible for the majority of drug-drug interactions and adverse drug reactions. A third group of cyts P450 are responsible for the biosynthesis or metabolism of essential endogenous compounds. This includes virtually all steroids (cholesterol, bile acids, estrogens, testosterone, cortisol, and vitamin D) and many lipids and eicosanoids. Cyts P450 exist in virtually every organ and tissue of humans. A second protein, cyt b5, modulates the biochemical mechanism and activity of selected cyts P450 in humans as well as animals. In fact, cyt b5 is essential for testosterone biosynthesis by a cyt P450 that is currently a therapeutic target for the treatment of prostate cancer. The knowledge gained from the proposed experiments will serve as the foundation for understanding the in vivo regulation of the catalytic mechanism of cyt P450. Although the cyt P450 interaction with cyt b5 has been intensely studied, significant gaps in our knowledge exist. The findings of the planned biochemical and structural experiments will also significantly enhance our ability to predict and eventually modify the routes of metabolism of a large number of environmental contaminants such as phthalates, bisphenol A, polychlorinated biphenyl (PCBs), and a vast number of currently used drugs, including chemotherapeutic agents, psychoactive compounds, and cardiovascular therapies. Knowledge of the molecular mechanism by which the activity of human cyts P450 can be modulated will also prove to be a tremendous asset in developing drugs and procedures to alter the large number of critical physiologic processes in which the 56 human cyts P450 participate. The results of the proposed studies will prove to be extremely valuable in designing less toxic and more specific therapeutic agents and prodrugs, especially chemotherapeutic agents and environmental contaminants. The proposed studies on how the activity of three of the most important human drug metabolizing cytochromes P450 is affected by a second protein, cytochrome b5, will provide significant new information, not readily available by any others means, about how the activity of human cytochromes P450 is regulated. The proposed studies will also provide a better understanding of the structure and function of, not only drug metabolizing cytochromes P450, but also of cytochromes P450 that are important in the metabolism of endogenous compounds such as testosterone, estrogens, and eicosanoids. This information about the chemical reactivity of human cyts P450 in the presence of its different redox partners will facilitate the design and development of new drugs and enhance the ability of investigators to genetically engineer cyts P450 in bacteria and other organisms to produce compounds to enhance human welfare
Keywords: (17Beta)-17-hydroxyandrost-4-en-3-one; 17-beta-Hydroxy-4-Androsten-3-one; 4, 4` isopropylidinediphenol; Active Sites; Aeroseb-HC; Affect; Anabolism; Androst-4-en-17beta-ol-3-one; Anesthestic Drugs; Anesthetic Agents; Anesthetic Drugs; Anesthetics; Animals; Bacteria; Bile Acids; Binding; Binding (Molecular Function); Biochemical; Biochemistry; Biodegradation; CYP3A4 protein, human; Cancer Causing Agents; Cancer of Prostate; Carcinogens; Cardiovascular; Cardiovascular Body System; Cardiovascular system; Cardiovascular system (all sites); Catalysis; Cetacort; Chemicals; Chemistry, Biological; Cholest-5-en-3-ol (3beta)-; Cholesterol; Clinical; Complex; Cort-Dome; Cortef; Cortenema; Cortisol; Cortispray; Cortril; Cytochrome P-450; Cytochrome P-450 Enzyme System; Cytochrome P450; Cytochrome P450 3A4; Cytochrome P450 Nifedipine Oxidase; Cytochrome P450, Subfamily IIIA (Niphedipine Oxidase), Polypeptide 3; Cytochrome P450, Subfamily IIIA (Niphedipine Oxidase), Polypeptide 4; Cytochrome P450, Subfamily IIIA, Polypeptide 4; Cytochrome P450PCN1; Cytochrome b-5; Cytochrome-B(5) Reductase; Cytochromes b5; Dehydrogenases; Delta4-androsten-17beta-ol-3-one; Dermacort; Development; Drug Interactions; Drug Precursors; Drug usage; Drugs; ENDOR; EPR spectroscopy; Eicosanoids; Eldecort; Electron Nuclear Double Resonance; Electron Paramagnetic Resonance; Electron Spin Resonance; Electron Spin Resonance Spectroscopy; Electronics; Electrons; Electrostatics; Employee Strikes; Engineering; Engineerings; Environmental Pollution; Estrogenic Agents; Estrogenic Compounds; Estrogens; Ferrate(2-), (7, 12-diethenyl-3, 8, 13, 17-tetramethyl-21H, 23H-porphine-2, 18-dipropanoato(4-)-N21, N22, N23, N24)-, dihydrogen, (SP-4-2)-; Ferricytochrome b5; Ferroprotoporphyrin; Foundations; Freezing; Genetic Alteration; Genetic Change; Genetic defect; Genetically Engineered Mouse; Glucocorticoid-Inducible P450; Goals; H+ element; Heme; Heme Iron; Heme b; Histidine; Histidine, L-isomer; Human; Human Activities; Human, General; Hydrocortisone; Hydrocortone; Hydrogen Ions; Hytone; In Vitro; Intermediary Metabolism; Investigators; Isoenzymes; Isozymes; Knowledge; L-Histidine; L-Phenylalanine; Laboratories; Life; Lipids; Liver; METBL; Malignant Tumor of the Prostate; Malignant neoplasm of prostate; Malignant prostatic tumor; Mammals, Mice; Man (Taxonomy); Man, Modern; Mechanics; Mediating; Medication; Metabolic Processes; Metabolism; Methods; Mice; Modeling; Modification; Molecular; Molecular Interaction; Mothers; Murine; Mus; Mutation; NADH-Cytochrome B5 Reductase; Nature; Negative Beta Particle; Negatrons; Nifedipine Oxidase; Nutracort; O element; O2 element; Oncogens; Organ; Organ System, Cardiovascular; Organism; Organism-Level Process; Organismal Process; Oxidases; Oxidation-Reduction; Oxidoreductase; Oxygen; P450; P450, FAMILY III; P450-3A4; P450-PCN1 (human); PCBs; Paramagnetic Resonance; Pathway interactions; Pharmaceutic Preparations; Pharmaceutical Preparations; Phenylalanine; Phenylalanine, L-Isomer; Physiologic Processes; Physiological Processes; Plants; Plants, General; Polychlorinated Biphenyls; Polychlorobiphenyl Compounds; Pregn-4-ene-3, 20-dione, 11, 17, 21-trihydroxy-, (11beta)-; Pro-Drugs; Procedures; Proctocort; Prodrugs; Property; Property, LOINC Axis 2; Prostate CA; Prostate Cancer; Prostatic Cancer; Proteins; Protoheme; Protoheme IX; Protons; Psychoactive Agents; Psychoactive Compound; Psychoactive Drugs; Psychopharmaceuticals; Psychotropic Drugs; Public Health; Quinine 3-Monooxygenase; Reaction; Redox; Reductases; Regulation; Relative; Relative (related person); Reporting; Research Personnel; Researchers; Role; Route; Series; Social Welfare; Spectroscopy, ESR; Steroid Compound; Steroid-Inducible P450- III; Steroids; Strikes; Strikes, Employee; Structure; Surface; Testosterone; Therapeutic Agents; Therapeutic Estrogen; Therapeutic Hydrocortisone; Therapeutic Testosterone; Toxic effect; Toxicities; Trans-Testosterone; VIT D; Vascular, Heart; Vitamin D; Xenobiotics; base; biosynthesis; bisphenol A; body system, hepatic; chemical reaction; chemotherapeutic agent; circulatory system; cyt P450 3A4; cytochrome P-450 3A4; cytochrome P-450 CYP3A4 (human); cytochrome P-450(PCN); cytochrome P-450(pcn1); cytochrome b5 reductase; design; designing; diphenylolpropane; drug use; drug/agent; electron paramagnetic resonance spectroscopy; environmental agent; environmental contaminant; environmental contamination; experiment; experimental research; experimental study; ferroheme; gene product; genome mutation; human tissue; in vitro activity; in vivo; living system; member; mutant; nifedipine oxidase, human; novel; organ system, hepatic; oxidation; oxidation reduction reaction; pathway; phthalates; polychlorobiphenyl; prevent; preventing; protonation; public health medicine (field); public health relevance; quinine 3-monooxygenase, human; research study; social role; testosterone biosynthesis; therapeutic target; tool; welfare
Relevance: RELEVANCE TO PUBLIC HEALTH: The proposed studies on how the activity of three of the most important human drug metabolizing cytochromes P450 is affected by a second protein, cytochrome b5, will provide significant new information, not readily available by any others means, about how the activity of human cytochromes P450 is regulated. The proposed studies will also provide a better understanding of the structure and function of, not only drug metabolizing cytochromes P450, but also of cytochromes P450 that are important in the metabolism of endogenous compounds such as testosterone, estrogens, and eicosanoids. This information about the chemical reactivity of human cyts P450 in the presence of its different redox partners will facilitate the design and development of new drugs and enhance the ability of investigators to genetically engineer cyts P450 in bacteria and other organisms to produce compounds to enhance human welfare
Project start date: 1985-07-01
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PA-07-070
5R01GM035533-22 (2010): $315000
Sponsored Links Excellgen http://Excellgen.com
PARTICIPATION OF CYTOCHROME B5 IN ANESTHETIC METABOLISM
Lucy A Waskell, Professor
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 5R01GM035533-08 from National Institute Of General Medical Sciences IRG: PHRA
Abstract: The long term objective of this proposal is to understand the mechanism by which drug metabolism and microsomal biosynthetic reactions modulate one another. The process occurs via an interaction of the two microsomal electron transport chains of which cytochrome P-450 and cytochrome b5 are components. Evidence exists that such interaction occurs in microsomes and in a reconstituted system employing purified enzymes. My laboratory has demonstrated that cytochrome b5 is required both in microsomes and in a purified reconstituted cytochrome P-450 system for the metabolism of the volatile anesthetic methoxyflurane. Preliminary data from my laboratory also indicate that methoxyflurane inhibits the synthesis of cholesterol, a substance which is known to require cytochrome b5 with unknown but possibly substantial in vivo consequences. The immediate goals of this project are three-fold. First, the role of cytochrome b5 in the metabolism of the volatile anesthetics, halothane, enflurane and isoflurane will be explored. The haloacid metabolites of the volatile anesthetics will be derivatized; the haloacid derivatives will be separated from interfering substances by gas chromatography and quantitated by mass spectrometry. The second objective is to elucidate the chemical basis of the inactivation of cytochrome b5 by the protein modifying agent, diethylpyrocarbonate. The experiments will be designed to first determine which amino acid(s) is modified and then determine the exact location of the amino acid in the protein. Our third and final short term aim is to determine the molecular basis for our observation that antibodies to cytochrome b5 inhibit methoxyflurane metabolism much more readily in phenobarbital induced liver than in lung microsomes. Cytochrome b5 and cytochrome P-450 LM2 from liver and lung will be purified, characterized and compared. Completion of the previously outlined experiments will significantly contribute to our knowledge of the factors which govern the interactions between cytochrome P-450 and cytochrome b5.
Keywords: anesthetic, cytochrome b, drug metabolism, microsome, NAD(P)H dehydrogenase, NADPH cytochrome c2 reductase, cholesterol, cyclic compound, cytochrome P450, cytochrome b5 reductase, electron transport, enflurane, haloacid, halothane, isoflurane, lysine, methoxyflurane, mixed function oxygenase, physical /chemical interaction, gas chromatography, laboratory rabbit, mass spectrometry
Project start date: 1985-07-01
Project end date: 1994-08-14
5R01GM035533-08 (1993): $209345
5R01GM035533-07 (1992): $198574
5R01GM035533-20 (2007): $294384
5R01GM035533-19 (2006): $296151
5R01GM035533-18 (2005): $296256
5R01GM035533-16 (2002): $277468
Sponsored Links Excellgen http://Excellgen.com
5R01GM035533-15 (2001): $267235
5R01GM035533-12 (1997): $216213
5R01GM035533-10 (1995): $252688
5R01GM035533-11 (1996): $244993
Grants awarded to Lucy A Waskell
WHY DO SUBSTRATES REQUIRE CYTOCHROME B5 FOR OXIDATION BY CYTOCHROME P450?
Lucy A Waskell, Professor
Institution:
Grant 5P41RR001081-190083 from National Center For Research Resources
Abstract: The overall long-term goal of our laboratory is to understand the molecular basis of the effect of cytochrome b5 (cyt b5) on the metabolism of certain substrates by cytochrome P-450 (cyt P-450), and to determine the physiological significance of this reaction. In particular, we are interested in elucidating the molecular basis of the marked stimulatory effect of cyt b5 on the metabolism of the volatile anesthetic, methoxyflurane, by cyt P-450 LM2 (2B4). These studies may eventually lead up the delineation of the etiology and pathophysiology of the postoperative hepatotoxicity attributed to the volatile anesthetics and to the development of safer anesthetics. These studies should also provide important information about the mechanism by which cyt P450 oxidizes its numerous endogenous and xenobiotic substrates. This knowledge should prove valuable in facilitating the design of clinically useful inhibitors of cytochrome P450 for therapeutic applications in fungal diseases, hyperaldosteronism, prostate cancer, benign prostatic hypertrophy and breast cancer. The more immediate short-term goal of this proposal is to use the 23,000 dalton cyt b5-cytochrome c (cyt c) complex as a model for the larger (72,000 dalton) hydrophobic cyt b5-cyt P450 complex and determine the structure of the model complex using high resolution NMR. The cytochrome b5-cytochrome c complex is a relevant model for the larger hydrophobic complex because cytochrome b5 uses approximately the same site to interact with both cytochrome c and cytochrome P450. Knowledge of the structure of the cyt b5-cyt ccomplex will provide insights, into 1) the mechanism, 2) pathway of electron transfer, between cyt b5 and its redox partners and 3) macromolecular recognition and protein dynamics. The appropriate three- and four- dimensional NMR experiments will be conducted on a 500 and 600 MHz NMR spectrometer with the available uniformly 13C/15N- labeled bovine cyt b5 in the presence and absence of cyt c. Completion of the proposed NMR experiments should provide us with important structural details about how certain amino acids of cyt b5 facilitate electron transfer with its redox partners, cyt P- 450, cyt b5 reductase and cyt c. The computing, graphics and programming facilities at the UCSF Computer Laboratory wll be used to construct three-dimensional molecular models based on results from our NMR studies, to display and examine these models, and to calculate the dynamics and interactions of the components in the models; these results should lead to a greater understanding of the structural basis for the requirement for cyt b5 for the metabolism of some substrates by cyt P-450.
WHY DO SUBSTRATES REQUIRE CYTOCHROME B5 FOR OXIDATION BY CYTOCHROME P450
Lucy A Waskell, Professor
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 5P41RR001081-220009 from National Center For Research Resources
Abstract: The overall long-term goal of our laboratory is to understand the molecular basis of the effect of cytochrome b5 (cyt b5) on the metabolism of certain substrates by cytochrome P-450 (cyt P-450) and to determine the physiological significance of this reaction. In particular, we are interested in elucidating the molecular basis of the marked stimulatory effect of cyt b5 on the metabolism of the volatile anesthetic, methoxyflurane, by cyt P-450 LM2 (2B4). These studies may eventually lead to the delineation of the etiology and pathophysiology of the postoperative hepatotoxicity attributed to the volatile anesthetics and to the development of safer anesthetics. These studies should also provide important information about the mechanism by which cyt P450 oxidizes its numerous endogenous and xenobiotic substrates. This knowledge should prove valuable in facilitating the design of clinically useful inhibitors of cytochrome P450 for therapeutic applications in fungal diseases, hyperaldosteronism, prostate cancer, benign prostatic hypertrophy and breast cancer. The more immediate short-term goal of this proposal is to use the 23,000 dalton cyt b5-cytochrome c (cyt c) complex as a model for the larger (72,000 dalton) hydrophobic cyt b5-cyt P450 complex and to determine the structure of the model complex using high resolution NMR. The cytochrome b5-cytochrome c complex is a relevant model for the larger hydrophobic complex because cytochrome b5 uses approximately the same site to interact with both cytochrome c and cytochrome P450. Knowledge of the structure of the cyt b5-cyt c complex will provide insights, into 1) the mechanism, 2) pathway of electron transfer, between cyt b5 and its redox partners, and 3) macromolecular recognition and protein dynamics. The appropriate three- and four-dimensional NMR experiments will be conducted on a 500 and 600 MHz NMR spectrometer with the available uniformly 13C/15N labeled bovine cyt b5 in the presence and absence of cyt c. Completion of the proposed NMR experiments should provide us with important structural details about how certain amino acids of cyt b5 facilitate electron transfer with its redox partners, cyt P-450, cyt b5 reductase and cyt c. The computing, graphics and programming facilities at the UCSF Computer Laboratory will be used to construct three-dimensional molecular models based on results from our NMR studies, to display and examine these models, and to calculate the dynamics and interactions of the components in the models; these results should lead to a greater understanding of the structural basis for the requirement for cyt b5 for the metabolism of some substrates by cyt P-450.
Keywords: biomedical resource, hematology, respiratory system
Participation Of Cytochrome B5 In Anesthetic Metabolism
Lucy A Waskell, Professor
University Of Michigan At Ann Arbor 3003 South State Street, Room 1040 Ann Arbor, Mi 481091274
Grant 2R01GM035533-17A2 from National Institute Of General Medical Sciences IRG: ALTX
Abstract: The cytochromes P450 are a ubiquitous superfamily of mixed function oxidases responsible for the oxidation of a vast number of compounds including the majority of drugs used by humans. In the course of our studies to determine how cytochrome b5 (cyt b5) stimulates the metabolism of substrates such as the anesthetic, methoxyflurane, by cytochrome P450 2B4 (cyt P450) we have recently measured, for the first time, the rate of reduction of a microsomal oxyferrous cyt P450, 2B4, by cyt P450 reductase. This experiment led to the discovery that catalysis by cyt P450 2B4 in the presence of cyt P450 reductase proceeds via a reduced oxyferrous intermediate. In striking contrast, catalysis by cyt P450 2134 in the presence of cyt b5 occurs rapidly without formation of a detectable intermediate allowing less time for unproductive side reactions to occur. The tools developed to measure the rate of reduction of oxyferrous cyt P450 2B4 will now be utilized to further probe the catalytic mechanism of cyt P450 and understand how cyt b5 enhances drug metabolism. The short term goals of this proposal are to elucidate the molecular origin and consequences of the strikingly different effects cyt b5 and cyt P450 reductase have on catalysis by cyt P450 2B4 while at the same time, gaining insight into the reaction mechanism of microsomal cyts P450. Aim 1. In order to advance our understanding of how cyt b5 and cyt P450 reductase exert their distinctive effects on cyt P450 2B4 catalysis we will trap and identify the intermediates formed during cyt P450 turnover using rapid freeze quench and cryoreduction EPR experiments in collaboration with Dr. Brian Hoffman. The time dependent formation of product during catalysis will be measured using rapid chemical quench techniques. Aim 2. Since the reduced oxyferrous intermediate which forms in the presence of wild type cyt P450 2B4 and cyt P450 reductase is short lived, three mutant cyts P450 2B4 (T302A, E301Q, F429H) which are expected to accumulate three different longer-lived reaction cycle intermediates [(Fe+3OOH)- (Fe+3OO)= (Fe+2OO)] will be isolated and studied to aid in the identification and characterization of the transient species. Aim 3. In an effort to gain a detailed molecular understanding of how cyt b5 expedites efficient catalysis by cyt P450 2B4, the amino acids forming interprotein contacts at the binding interface between cyt P450 2B4 and cyt b5 will be identified using site-directed mutagenesis and double-mutant cycle experiments.
Keywords: anesthetic, cytochrome P450, cytochrome b, drug metabolism, enzyme mechanism, cytochrome b5 reductase, enzyme activity, enzyme substrate, gene mutation, methoxyflurane, electron spin resonance spectroscopy, site directed mutagenesis, stop flow technique
Project start date: 1985-07-01
Project end date: 2008-03-31
2R01GM035533-17A2 (2004): $404736
2R01GM035533-14A2 (2000): $278901
PARTICIPATION IN CYTOCHROME B5 IN ANESTHETIC METABOLISM
Lucy A Waskell, Professor
University Of Michigan At Ann Arbor, 1040 Wolverine Tower, Ann Arbor, Mi 48109-1274
Grant 2R01GM035533-21A2 from National Institute Of General Medical Sciences
Abstract: The cytochromes P450 (cyts P450) are a ubiquitous superfamily of oxidases whose activity is influenced by a second protein, cytochrome b5 (cyt b5). The long-term goal of this project is to understand the structural and mechanistic basis of the modification of human cyt P450-mediated metabolism by cyt b5 in order to develop and employ strategies to modify the activity of these essential isozymes for human benefit. In order to understand the molecular mechanism by which cyt b5 influences oxidation by cyt P450 in vivo, one must understand the details of how cyt b5 interacts with cyt P450 and how it modifies the catalytic cycle of cyt P450 relative to cyt P450 reductase. There are more than 8,000 members of the cyts P450 superfamily. They are found in all kingdoms of living organisms and plants and are referred to as Mother Nature´s blowtorch, due to their ability to oxidize a vast number of stable chemical entities. Humans possess 56 different cyts P450, many of which are essential for early development and life itself. Other human cyts P450 determine the toxicity, duration of action, and elimination of the vast majority of therapeutic agents, carcinogens, and environmental agents to which humans are exposed. Xenobiotic metabolizing cyts P450 are also responsible for the majority of drug-drug interactions and adverse drug reactions. A third group of cyts P450 are responsible for the biosynthesis or metabolism of essential endogenous compounds. This includes virtually all steroids (cholesterol, bile acids, estrogens, testosterone, cortisol, and vitamin D) and many lipids and eicosanoids. Cyts P450 exist in virtually every organ and tissue of humans. A second protein, cyt b5, modulates the biochemical mechanism and activity of selected cyts P450 in humans as well as animals. In fact, cyt b5 is essential for testosterone biosynthesis by a cyt P450 that is currently a therapeutic target for the treatment of prostate cancer. The knowledge gained from the proposed experiments will serve as the foundation for understanding the in vivo regulation of the catalytic mechanism of cyt P450. Although the cyt P450 interaction with cyt b5 has been intensely studied, significant gaps in our knowledge exist. The findings of the planned biochemical and structural experiments will also significantly enhance our ability to predict and eventually modify the routes of metabolism of a large number of environmental contaminants such as phthalates, bisphenol A, polychlorinated biphenyl (PCBs), and a vast number of currently used drugs, including chemotherapeutic agents, psychoactive compounds, and cardiovascular therapies. Knowledge of the molecular mechanism by which the activity of human cyts P450 can be modulated will also prove to be a tremendous asset in developing drugs and procedures to alter the large number of critical physiologic processes in which the 56 human cyts P450 participate. The results of the proposed studies will prove to be extremely valuable in designing less toxic and more specific therapeutic agents and prodrugs, especially chemotherapeutic agents and environmental contaminants. The proposed studies on how the activity of three of the most important human drug metabolizing cytochromes P450 is affected by a second protein, cytochrome b5, will provide significant new information, not readily available by any others means, about how the activity of human cytochromes P450 is regulated. The proposed studies will also provide a better understanding of the structure and function of, not only drug metabolizing cytochromes P450, but also of cytochromes P450 that are important in the metabolism of endogenous compounds such as testosterone, estrogens, and eicosanoids. This information about the chemical reactivity of human cyts P450 in the presence of its different redox partners will facilitate the design and development of new drugs and enhance the ability of investigators to genetically engineer cyts P450 in bacteria and other organisms to produce compounds to enhance human welfare
Keywords: (17Beta)-17-hydroxyandrost-4-en-3-one; 17-beta-Hydroxy-4-Androsten-3-one; 4, 4` isopropylidinediphenol; Acids, Bile; Active Sites; Aeroseb-HC; Affect; Anabolism; Androst-4-en-17beta-ol-3-one; Anesthestic Drugs; Anesthetic Agents; Anesthetic Drugs; Anesthetics; Animals; Bacteria; Bile Acids; Binding; Binding (Molecular Function); Biochemical; Biochemistry; Biodegradation; CYP3A4 protein, human; Cancer Causing Agents; Cancer of Prostate; Carcinogens; Cardiovascular; Cardiovascular Body System; Cardiovascular system; Cardiovascular system (all sites); Catalysis; Cetacort; Chemicals; Chemistry, Biological; Cholest-5-en-3-ol (3beta)-; Cholesterol; Clinical; Complex; Cort-Dome; Cortef; Cortenema; Cortisol; Cortispray; Cortril; Cytochrome P-450; Cytochrome P-450 Enzyme System; Cytochrome P450; Cytochrome P450 3A4; Cytochrome P450 Nifedipine Oxidase; Cytochrome P450, Subfamily IIIA (Niphedipine Oxidase), Polypeptide 3; Cytochrome P450, Subfamily IIIA (Niphedipine Oxidase), Polypeptide 4; Cytochrome P450, Subfamily IIIA, Polypeptide 4; Cytochrome P450PCN1; Cytochrome b-5; Cytochrome-B(5) Reductase; Cytochromes b5; Dehydrogenases; Delta4-androsten-17beta-ol-3-one; Dermacort; Development; Drug Interactions; Drug Precursors; Drug usage; Drugs; ENDOR; EPR spectroscopy; Eicosanoids; Eldecort; Electron Nuclear Double Resonance; Electron Paramagnetic Resonance; Electron Spin Resonance; Electron Spin Resonance Spectroscopy; Electronics; Electrons; Electrostatics; Employee Strikes; Engineering; Engineerings; Environmental Pollution; Estrogenic Agents; Estrogenic Compounds; Estrogens; Ferrate(2-), (7, 12-diethenyl-3, 8, 13, 17-tetramethyl-21H, 23H-porphine-2, 18-dipropanoato(4-)-N21, N22, N23, N24)-, dihydrogen, (SP-4-2)-; Ferricytochrome b5; Ferroprotoporphyrin; Foundations; Freezing; Genetic Alteration; Genetic Change; Genetic defect; Genetically Engineered Mouse; Glucocorticoid-Inducible P450; Goals; H+ element; Heme; Heme Iron; Heme b; Histidine; Histidine, L-isomer; Human; Human Activities; Human, General; Hydrocortisone; Hydrocortone; Hydrogen Ions; Hytone; In Vitro; Intermediary Metabolism; Investigators; Isoenzymes; Isozymes; Knowledge; L-Histidine; L-Phenylalanine; Laboratories; Life; Lipids; Liver; METBL; Malignant Tumor of the Prostate; Malignant neoplasm of prostate; Malignant prostatic tumor; Mammals, Mice; Man (Taxonomy); Man, Modern; Mechanics; Mediating; Medication; Metabolic Processes; Metabolism; Methods; Mice; Modeling; Modification; Molecular; Molecular Interaction; Mothers; Murine; Mus; Mutation; NADH-Cytochrome B5 Reductase; Nature; Negative Beta Particle; Negatrons; Nifedipine Oxidase; Nutracort; O element; O2 element; Oncogens; Organ; Organ System, Cardiovascular; Organism; Organism-Level Process; Organismal Process; Oxidases; Oxidation-Reduction; Oxidoreductase; Oxygen; P450; P450, FAMILY III; P450-3A4; P450-PCN1 (human); PCBs; Paramagnetic Resonance; Pathway interactions; Pharmaceutic Preparations; Pharmaceutical Preparations; Phenylalanine; Phenylalanine, L-Isomer; Physiologic Processes; Physiological Processes; Plants; Plants, General; Polychlorinated Biphenyls; Polychlorobiphenyl Compounds; Pregn-4-ene-3, 20-dione, 11, 17, 21-trihydroxy-, (11beta)-; Pro-Drugs; Procedures; Proctocort; Prodrugs; Property; Property, LOINC Axis 2; Prostate CA; Prostate Cancer; Prostatic Cancer; Proteins; Protoheme; Protoheme IX; Protons; Psychoactive Agents; Psychoactive Compound; Psychoactive Drugs; Psychopharmaceuticals; Psychotropic Drugs; Public Health; Quinine 3-Monooxygenase; Reaction; Redox; Reductases; Regulation; Relative; Relative (related person); Reporting; Research Personnel; Researchers; Role; Route; Series; Sites, Active; Social Welfare; Spectroscopy, ESR; Steroid Compound; Steroid-Inducible P450- III; Steroids; Strikes; Strikes, Employee; Structure; Surface; Testosterone; Therapeutic Agents; Therapeutic Estrogen; Therapeutic Hydrocortisone; Therapeutic Testosterone; Toxic effect; Toxicities; Trans-Testosterone; VIT D; Vascular, Heart; Vitamin D; Xenobiotics; base; biosynthesis; bisphenol A; body system, hepatic; chemical reaction; chemotherapeutic agent; circulatory system; cyt P450 3A4; cytochrome P-450 3A4; cytochrome P-450 CYP3A4 (human); cytochrome P-450(PCN); cytochrome P-450(pcn1); cytochrome b5 reductase; design; designing; diphenylolpropane; drug use; drug/agent; electron paramagnetic resonance spectroscopy; environmental agent; environmental contaminant; environmental contamination; experiment; experimental research; experimental study; ferroheme; gene product; genome mutation; human tissue; in vitro activity; in vivo; living system; member; mutant; nifedipine oxidase, human; novel; organ system, hepatic; oxidation; oxidation reduction reaction; pathway; phthalates; polychlorobiphenyl; prevent; preventing; protonation; public health medicine (field); public health relevance; quinine 3-monooxygenase, human; research study; social role; testosterone biosynthesis; therapeutic target; tool; welfare
Relevance: RELEVANCE TO PUBLIC HEALTH: The proposed studies on how the activity of three of the most important human drug metabolizing cytochromes P450 is affected by a second protein, cytochrome b5, will provide significant new information, not readily available by any others means, about how the activity of human cytochromes P450 is regulated. The proposed studies will also provide a better understanding of the structure and function of, not only drug metabolizing cytochromes P450, but also of cytochromes P450 that are important in the metabolism of endogenous compounds such as testosterone, estrogens, and eicosanoids. This information about the chemical reactivity of human cyts P450 in the presence of its different redox partners will facilitate the design and development of new drugs and enhance the ability of investigators to genetically engineer cyts P450 in bacteria and other organisms to produce compounds to enhance human welfare
Project start date: 1985-07-01
Project end date: 2011-08-31
Budget start date: 30-SEP-2009
Budget end date: 31-AUG-2010
PFA/PA: PA-07-070
2R01GM035533-21A2 (2009): $307948
PARTICIPATION OF CYTOCHROME B5 IN ANESTHETIC METABOLISM
Lucy A Waskell, Professor
Anesthesiauniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962
Grant 2R01GM035533-09A1 from National Institute Of General Medical Sciences IRG: PHRA
Abstract: Cyt 5 can increase, decrease or have no effect on substrate metabolism by cyt P450. How and why cyt b5 can have such an unpredictable effect on cyt P450 catalyzed oxidations has puzzled researchers for decades. With the recent marked increase in the number of endogenous compounds and drugs that have been shown to increase their metabolism in the presence of cyt b5, this question is becoming more interesting and biologically relevant. The long term objective of this proposal is to understand the molecular basis of the marked stimulation of the cyt P450 catalyzed metabolism of certain substrates by cyt b5 and to determine the physiological significance of this reaction. The problem will be addressed by elucidating the mechanism by which the model compound methoxyflurane (MF), a volatile anesthetic, induces the requirement for cyt b5 for its metabolism by cyt P450 2B4 (LM2). These studies may eventually lead to the delineation of the etiology and pathophysiology of the postoperative hepatotoxicity attributed to the volatile anesthetics, and should contribute to our understanding of the mechanism of oxidation by cyt P450. The short term goals of this proposal are three-fold. The first specific aim, using stopped-flow spectrophotometry, is to investigate the effect of the substrates, MF and benzphetamine on the binding of cyt P450 reductase and cyt b5 to oxyferrous cyt P450 and on the rate of reduction of oxyferrous cyt P450 by these two proteins. The rate at which the product of both substrates is released from the substrate binding site will also be investigated. The interpretation of these studies will be greatly facilitated by calculations of the spectra, electronic structure and spin distribution of stable and transient intermediates in the cyt P450 reaction cycle, which will be performed in collaboration with Dr. Gilda Loew. The second specific aim is to continue our mutation studies into the role of specific amino acids of cyt b5 in the oxidation of MF by cyt P450. Of major interest will be two groups of residues, the acidic residues around the heme of cyt b5 which are involved in binding to its soluble redox partners, and the amino acids on the putative inter-protein binding surface. The third specific aim will be to locate the interprotein binding site on cyt P450 for cyt b5. This will be accomplished using a model of cyt P450 2B4 to select which amino acids on the surface of cyt P450 2B4 might be involved in interactions with cyt b5. The selected amino acids will then be systematically mutated to alanine using the technique of alanine scanning. The ability of the mutant cyt P450 molecules to bind cyt b5 and to function in electron transfer with these proteins will be evaluated
Keywords: anesthetic, cytochrome P450, cytochrome b, drug metabolism, enzyme mechanism active site, alanine, cytochrome b5 reductase, electron transport, enzyme activity, enzyme substrate, methoxyflurane, mixed function oxygenase laboratory rabbit, site directed mutagenesis, spectrometry, stoichiometry, stop flow technique
Project start date: 1985-07-01
Project end date: 1998-07-31
2R01GM035533-09A1 (1994): $204441