Cytochrome P450

ContributorsE. Anne,, W.L. Backes, I. Benveniste, R. Bernhardt,L.S. Birnbaum, S.D. Black, J. Blanck, E. Bresnick, M.J. Coon,CJ. Corbin, K.M. Curnow, A.K. Daly, X. Ding, J. Doehmer,F. Durst, M.J. Fasco, R. Feyereisen, Y. Fujii-Kuriyama, Y. Funae,F.J. Gonzalez, S.E. Graham-Lorence, R.W. Gray, H. Greim,F.P. Guengerich, J.-A. Gustafsson, D.K. Hammond, B.W. Hollis,J.R. Idle, S. Imaoka, I. Jansson, C. Jung, O. Kappeli,L.S. Kaminsky, H. Kappus, C.B. Kasper, R. Kato, M.W. Kilgore,W.N. Kiihn-Velten, W. Levin, M.S. Mahendroo, M.A. Marietta,G.D. Means, C.R. Mendelson, D. Muller-Enoch, M. Noshiro,R.S. Obach, T. Ogishima, K. Okuda, T. Omura, P.R. Ortiz deMontellano, L. Pascoe, H. Rein, K. Ruckpaul, D.E. Ryan,D. Sanglard, J.B. Schenkman, L.R. Schwarz, A.L. Shen,E.R. Simpson, J.F. Sinclair, P.R. Sinclair, K. Sogawa,J.J. Stegeman, D.J. Stralka, H.W. Strobel, G. Strobl,L.E. Vickery, M. Warner, D.J. Waxman, K.A. White P.C. White,T. White, F.J. Wiebel, K. Wikvall, T. Wolff, Y. Yamazoe,Y. Yoshida, P. Zimniak

Editors

John B. Schenkman and Helmut Greim

Springer-VerlagBerlin Heidelberg New York London ParisTokyo Hong Kong Barcelona Budapest

Contents

Section I: The Monooxygenase System

CHAPTER 1

Historical Background and Description of the Cytochrome P450Monooxygenase SystemJ.B. SCHENKMAN. With 1 Figure 3

A. Historical Introduction 3B. Cytochrome P450 4

I. Multiple Forms 4II. Hemoprotein Characteristics 5

HI. Oxygen Activation 6IV. Uncoupling of Monooxygenations 7V. Role for Cytochrome b5 8

C. Molecular Biology of Cytochrome P450 8References .- 10

CHAPTER 2

NADPH-Cytochrome P450 Reductase: FunctionW.L. BACKES. With 4 Figures 15

A. Introduction 15B. Early Characterization Studies 15

I. Identification of Flavins 15II. Identification of Physiological Electron Acceptor 16

III. Purification of NADPH-Cytochrome c Reductase 16C. Reduction of NADPH-Cytochrome P450 Reductase 17

I. Identification of Air-Stable Semiquinone 17II. Function of Flavins in Reductase Reduction 18

HI. Kinetics of NADPH-Cytochrome P450 Reductase Reduction .. 211. Interflavin Electron Transfers 222. Mechanistic Details of Reductase Reduction 23

D. Electron Transfer to Cytochrome P450 and Other ElectronCarriers 24

XIV Contents

I. General Characteristics of Cytochrome P450-DependentReactions 24

II. Electron Transfer to Cytochrome P450 261. Rate of Transfer from Different Reductase Reduction

States 262. Electron Shuttling During the Monooxygenase Reaction . . . 27

III. NADPH-Cytochrome P450 Reductase Involvement in OtherReactions 29

References 30

CHAPTER 3

Protein and Gene Structure and Regulation of NADPH-CytochromeP450 OxidoreductaseA.L. SHEN and C.B. KASPER. With 6 Figures 35

A. Introduction 35B. Structure of NADPH-Cytochrome P450 Oxidoreductase 37

I. Membrane-Binding Domain 37II. Flavin Mononucleotide-Binding Domain 37

1. Binding of the FMN Phosphate Group 372. Binding of the FMN Isoalloxazine 38

III. Substrate-Binding Domain 391. Chemical Modification, Cross-Linking, and Site-Directed

Mutagenesis 402. Chemical Modification and Site-Directed Mutagenesis of

Cytochrome P450 42IV. Flavin Adenine Dinucleotide-Binding Domain 42V. NADPH-Binding Domain 44

1. Dinucleotide-Binding Site 442. Role of Cysteine in NADPH Binding 453. Binding of 2'-Phosphate of NADPH 46

VI. Interactions Between Domains *.. 46C. Structure of the NADPH-Cytochrome P450 Oxidoreductase Gene . 48D. Regulation of NADPH-Cytochrome P450 Oxidoreductase Gene

Expression 50I. Induction 50

II. Developmental Regulation 51References 52

CHAPTER 4

Localization of Cytochrome P450 in Membranes: MitochondriaT. OMURA. With 2 Figures 61

A. Cytochrome P450 in Mitochondria 61

Contents XV

B. Topology of Cytochrome P450 Molecules in the MitochondrialInner Membrane : 62

C. Biosynthesis of Mitochondrial Cytochrome P450s as PrecursorForms 62

D. Import of Cytochrome P450 Precursors into Mitochondria andTheir Processing to Mature Membrane-Bound Forms 65

References 67

CHAPTER 5

Localization of Cytochrome P450 in Membranes: Reconstituted SystemsD. MULLER-ENOCH. With 3 Figures 71

A. Introduction 71B. Soluble Reconstituted Systems 72

I. Formation of the Catalytically Active Cytochrome P450:Reductase Complex 721. Binary Complex Formation 722. Dissociation of the Preformed Cytochrome P450: Reductase

Complex 743. Association of the Cytochrome P450: Reductase Complex . . 764. Reconstitution of Maximum Cytochrome P450 Supported

Activity Without Lipid 77II. Effects on the Reconstituted Soluble Cytochrome P450:

Reductase System 781. Detergent-Mediated Effects 782. Organic Solvent-Mediated Effects 793. Effects of Cytochrome b5 794. Self-Association of Monomers 805. Electrostatic Interactions Between Cytochrome P450 and

Reductase 80C. Vesicular Reconstituted Systems 81D. Conclusions 82References 83

Section II: The Monooxygenase Reactions

CHAPTER 6

Metabolic Reactions: Types of Reactions of Cytochrome P450 EnzymesF.P. GUENGERICH. With 1 Figure 89

A. Introduction 89B. General Features of Cytochrome P450 Catalysis 89C. Specific Oxidative Reactions 91

XVI Contents

I. Carbon Hydroxylation 91II. Heteroatom Oxygenation 91

III. Heteroatom Release 93IV. Rearrangements Related to Heteroatom Oxidations 94V. Oxidations of 7r-Systems 96

VI. Reactions Involving Hypervalent Oxygen Substrates 98D. Reductive Reactions 99E. Conclusions 100References 100

CHAPTER 7

Metabolic Reactions: Mechanisms of Substrate OxygenationH. REIN and C. JUNG. With 1 Figure 105

A. Introduction 105B. Substrate Interaction with Cytochrome P450 106

I. Relationship Between Spectral Changes and Spin State 106II. Substrate-Induced Spin State Equilibrium Shift 108

III. Substrate-Induced Redox Potential Shift 110C. Reduction Control by the Spin/Redox Couple I l lD. Ternary Cytochrome P450-Dioxygen-Substrate Complex 112E. Cleavage of the Dioxygen Bond 113F. Regulation of Substrate Turnover 115References 116

CHAPTER 8

Liver Cytochrome P450 Metabolism of Endogenous Steroid Hormones,Bile Acids, and Fatty AcidsP. ZIMNIAK and D.J. WAXMAN. With 5 Figures 123

A. Introduction 123B. Hydroxylation of Neutral Steroids (Steroid Hormones) by Liver

Cytochromes P450 125I. Substrate Specificity 125

II. Developmental and Hormonal Regulation 127III. Significance of Steroid Hydroxylations 128

C. Hydroxylations of Bile Acids 129I. Bile Acid Biosynthesis: Physiological Implications of Bile Acid

Hydrophobicity 129II. Bile Acid Hydroxylase Cytochromes P450 132

D. Hydroxylations of Fatty Acids 134E. Oxidation of Ethanol and Other Low Molecular Weight

Compounds 136

Contents XVII

F. Cytochrome P450 Mediated Endogenous Metabolism in OtherSystems 137

G. Conclusion 138References 139

CHAPTER 9

Metabolic Reactions: Role of Cytochrome P450 in the Formation ofReactive Oxygen SpeciesH. KAPPUS. With 2 Figures 145

A. Definition of Reactive Oxygen 145B. Reactive Oxygen Generation During Drug Metabolism 147C. Formation of Drug Radicals During Metabolism by Cytochrome

P450 150D. Lipid Peroxidation Catalyzed by Cytochrome P450 151E. Conclusions 152References 152

CHAPTER 10

Cytochrome P450 Structure and FunctionS.D. BLACK. With 1 Figure 155

A. Introduction 155B. Analysis of the Tertiary Structure of Cytochrome P450cam 156C. Approaches to Computation of Three-Dimensional Models of

Cytochromes P450 159I. General Approach 159

II. Examples of Three-Dimensional Protein Models 162III. Three-Dimensional Models of Cytochromes P450 162IV. Limitations 165

D. Problems Addressable with Three-Dimensional Models ofCytochromes P450. 165

References 166

CHAPTER 11

Structure of Cytochrome P450: Heme-Binding Site and Heme ReactivityP.R. ORTIZ DE MONTELLANO and S.E. GRAHAM-LORENCE.

With 4 Figures 169

A. Introduction 169B. Cytochrome P450cam (CYP101) 169C. Sequence Alignments 170

XVIII Contents

D. Mutagenesis Studies 170E. Topological Analysis by Covalent Heme Modification 173

I. Terminal Olefins and Acetylenes 173II. Phenylhydrazine and Phenyldiazene 174

III. 4-Alkyl-l,4-dihydropyridines 177F. Topological Information from Substrate Specificity 178

I. Cytochrome P4501A1 (CYP1A1) 178II. Cytochrome P4502D6 (CYP2D6) 178

G. Conclusions 178References 179

CHAPTER 12

Cytochrome P450: Probes of Active Site ResiduesL.S. KAMINSKY, R.S. OBACH, and M.J. FASCO. With 4 Figures 183

A. Introduction 183B. Substrate-Binding Site Probes 184

I. Substrate-Binding Site-Directed Probes 1841. Affinity and Photoaffinity Probes 1842. Suicide Substrates 185

II. Amino Acid Residue Modifications 185III. Substrate Regio- and Stereoselectivity 188IV. Comparisons of Cytochrome P450 Primary Structures 191

C. Conclusion 191References 192

CHAPTER 13

Structural Models for Substrates and Inhibitors of Cytochrome P450EnzymesT. WOLFF, G. STROBL, and H. GREIM. With 2 Figures 195

A. Introduction 195B. Three-Dimensional Structure of the Active Site as a Basis for

Substrate and Inhibitor Design 195I. X-Ray Structure of Cytochrome P450cam 195

II. "Homology Building" of the Active Site of MammalianEnzymes 197

C. Approaches to Design Substrate and Inhibitor Models 197I. Empirical Models 197

II. Computer-Aided Molecular Design of Pharmacophor Models . . 2001. Quantitative Structure Activity Relationship (QSAR)

Analysis 2002. Molecular Modeling 2023. Molecular Modeling of Substrates and Inhibitors of

Cytochrome P450 2D6 202

Contents XIX

D. Conclusions 205

References 205

Section III: Forms of Microsomal P450

CHAPTER 14Cytochrome P450 Evolution and NomenclatureF.J. GONZALEZ 211

A. Evolution of Cytochromes P450 211B. Cytochrome P450 Nomenclature 216

I. Definitions of Families and Subfamilies 216II. Orthologous Genes 217

III. Allelic Variants 217IV. The Cytochrome P450 Superfamily 218

References 218

CHAPTER 15

Cytochrome P450 in RodentsY. FUNAE and S. IMAOKA 221

A. Introduction 221B. Rat Cytochrome P450 Forms 221

I. Forms Isolated 2211. CYP1A1, 1A2 2212. CYP2A1, 2A2 2243. CYP2B1, 2B2 2244. CYP2C6, 2C7, 2C11, 2C12, 2C13 2245. CYP2D1,2D2 2256. CYP2E1 2257. CYP3A1, 3A2 2258. CYP4A1, 4A2, 4A3 2269. Other Forms of Cytochrome P450 226

II. Catalytic Properties of Rat Cytochrome P450 226HI. Changes in Rat Hepatic Cytochrome P450 by Treatment with

Chemicals and Under Different PathophysiologicalConditions 228

C. Rabbit and Mouse Cytochromes P450 229References 233

CHAPTER 16

Cytochrome P450 in HumansF.J. GONZALEZ 239

A. Introduction 239B. CYP1A Subfamily 240

XX Contents

I. CYP1A1 240II. CYP1A2 241

C. CYP2A Subfamily 242D. CYP2B Subfamily 244E. CYP2C Subfamily 244F. CYP2D Subfamily 246G. CYP2E Subfamily 246H. CYP2F Subfamily 248I. CYP3A Subfamily 248References 249

CHAPTER 17

Avian Cytochrome P450J.F. SINCLAIR and P.R. SINCLAIR 259

A. Introduction 259B. Phenobarbital-Inducible Cytochrome P450 in Avians 259

I. Historical Perspective 259II. Hepatic Mixed Function Oxidase Activities Induced by

Phenobarbital Treatment in Avians 2601. Enzyme Activities 2602. Embryonic Response 260

III. Identification of Phenobarbital-Inducible Forms of CytochromeP450 2611. Cytochrome P450 2H1 and 2H2 ; 2612. Other Phenobarbital-Induced Forms of Cytochrome P450 . . 261

IV. Mechanism of Induction of Cytochrome P450 2H1/2 2621. Properties of Inducers 2622. Expression of Cytochrome P450 2H1/2 Protein and mRNAs 2623. Role of Heme in Expression of Cytochrome P450 2H1/2 . . . 2634. Effect of Protein Synthesis Inhibitors on Expression of

Cytochrome P450 2H1/2 2635. Mechanism of Coordinate Induction of Cytochrome P450

and ALAS 264C. Planar Polycyclic Aromatic Hydrocarbon-Inducible Cytochrome

P450 in Avians 265I. Historical Perspective 265

II. Hepatic Mixed Function Oxidase Activities Induced by PlanarPolycyclic Aromatic Hydrocarbons in Avians 2661. Enzyme Activities 266

III. Identification of Planar Polycyclic Aromatic-Inducible Forms ofAvian Cytochrome P450 266

IV. Mechanism of Induction of Cytochrome P4501A 267D. Acetone-and Alcohol-Inducible Cytochrome P450 in Avians 269

Contents XXI

I. Hepatic Mixed Function Oxidase Activities Induced byAcetone or Ethanol in Avians 269

II. Identification of Acetone-Inducible Forms of Cytochrome P450 269E. Additional Avian Forms of Cytochrome P450 270

I. Dexamethasone-Inducible Cytochrome P450 270II. Vitamin D Hydroxylase 271

III. Steroid Metabolism 271F. Conclusions 271References 272

CHAPTER 18

Cytochrome P450 Forms in FishJ.J. STEGEMAN. With 1 Figure 279

A. Introduction 279B. Microsomal Cytochrome P450 in Fish 280C. Cytochrome P450 Forms in Liver 281D. Cytochrome P450 Form Relationships 283

I. Gene Family 1 2831. Subfamily 1A 2832. Multiple 1A Genes? 2833. Cytochrome P450 1A Regulation 284

II. Gene Family 2 2851. Subfamily 2B .2852. Subfamily 2E 286

III. Gene Family 3 286E. Extrahepatic Cytochrome P450 286

I. Cytochrome P450 1A (Distribution) 286II. Cytochrome P450LM2/KM2 287

F. Conclusion 287References 288

CHAPTER 19

Cytochrome P450 in PlantsF. DURST and I. BENVENISTE. With 4 Figures 293

A. Introduction 293B. Components and Distribution 293C. Forms of Cytochrome P450 295

I. Physiological Substrates 2961. Phenylpropanoids 2962. Terpenes 2963. Fatty Acids 297

XXII Contents

4. Cyanogenic Glucosides 300II. Metabolism of Xenobiotics 305

D. Conclusion 306References 306

CHAPTER 20

Cytochrome P450 in InsectsR. FEYEREISEN 311

A. Introduction 311B. Structures of Insect Cytochrome P450 311

I. CYP6A1 312II. CYP6A2 312

III. CYP6B1 313IV. CYP4C1 • • • • 313V. CYP4D1 313

VI. NADPH Cytochrome P450 Reductase 314C. Functions of Insect Cytochrome P450 314

I. Metabolism of Foreign Compounds 314II. Ecdysteroid Metabolism 315

1. Ecdysone 20-Monooxygenase 3152. Biosynthesis of Ecdysone Precursors 317

III. Juvenile Hormone Biosynthesis 318IV. Pheromone Biosynthesis 318V. Fatty Acid Metabolism 318

D. Cytochrome P450 in Drosophila melanogaster 319E. Insect Systems for the Expression of Cytochrome P450 Genes 320

I. Baculovirus 320II. P-Element Transformation of Drosophila 320

References 321

CHAPTER 21

Cytochrome P450 in Unicellular OrganismsD. SANGLARD and O. KAPPELI 325

A. Introduction 325B. Cytochromes P450 from Bacteria 325

I. Camphor-Hydroxylating Cytochrome P450 from Pseudomonasputida 327

II. Cytochrome P450 Linalool 8-Methyl Hydroxylase fromPseudomonas putida (icognita) 329

III. Cytochrome P450 Fatty Hydroxylase from Bacillusmegaterium 329

Contents XXIII

IV. Cytochromes P450 from Actinomycetes 3301. Herbicide-Inducible Cytochromes P450 from Streptomyces

grisoleus 3312. Soybean Flour-Induced Cytrochrome P450 from

Streptomyces griseus 3313. Cytochrome P450 6-Deoxyerythronolide B Hydroxylase

from Saccharopolyspora erythracea 3324. Compactin-Inducible Cytochrome P450 Hydroxylase from

Streptomyces carbophilus 3335. Veratrole-Inducible Cytochrome P450 from Streptomyces

setonii 333C. Cytochromes P450 from Eukaryotic Microorganisms 334

I. Cytochromes P450 from Yeasts 3341. Cytochrome P450 14a-Lanosterol Demethylase 3342. Alkane-Inducible Cytochrome P450 Monooxygenases 3373. Cytochrome P450 Involved in Spore Wall Maturation 338

II. Cytochromes P450 from Fungi 3381. Cytochrome P450 Benzoate-p-hydroxylase from Aspergillus

niger 3382. Cytochrome P450 Pisatin Demethylase from Nectria

haematococca 3403. Cytochrome P450 Monooxygenases from Fusarium

oxysporium 3404. 1 la-Progesterone Hydroxylase from Rhizopium nigricans .. 3415. Cycloheximide-Inducible Cytochrome P450 from

Neurospora crassa 3416. Cytochrome P450 Monooxygenases from Cunninghamella

Species 341D. Conclusions 342References 343

CHAPTER 22

Extrahepatic Microsomal Forms: Olfactory Cytochrome P450X. DING and M. J. COON. With 1 Figure 351

A. Introduction 351B. Olfactory Cytochrome P450 of Rabbits 352

I. NMa and NMb 352II. Other Forms 355

C. Olfactory Cytochrome P450 in Other Species 356I. Cytochrome P450olfl and Cytochrome P450olf2 356

II. Other Forms 357D. Future Prospects 358References 358

XXIV Contents

CHAPTER 23

Extrahepatic Microsomal Forms: Gastrointestinal Cytochromes P450,Assessment and EvaluationH.W. STROBEL, D.J. STRALKA, D.K. HAMMOND, and T. WHITE. With 3Figures 363

A. Introduction 363B. Human Colon Drug Metabolism 364

I. Microsomal Activities 364II. Cytochrome P450 365

C. Human Colon Tumor Cell Drug Metabolism 366I. Microsomal Activities 366

II. Intact Cell Assays 367III. Cytochromes P450 368

D. Discussion 369References 370

CHAPTER 24

Extrahepatic Microsomal Forms: Lung Microsomal Cytochrome P450IsozymesE. ARIN? 373

A. Introduction 373B. Lung Microsomal Cytochrome P450 Dependent Monooxygenases .. 373

I. Species Differences in Lung Cytochrome P450 DependentMonooxygenase,Activities 373

C. Lung Microsomal Cytochrome P450 Isozymes 374I. Rabbit Lung Microsomal Cytochromes P450 374

1. Properties and Regulation of Rabbit Lung CytochromeP4502B4 (P450LgM2) 375

2. Comparison of Properties of Sheep and Rabbit LungCytochrome P450LgM2 376

3. Properties and Regulation of Rabbit Lung CytochromeP4504B1 (P450LgM5) 377

4. Properties and Regulation of Rabbit Lung CytochromeP450LgM6 (P4501A1) 378

5. Properties and Regulation of Rabbit Lung CytochromesP450PGW, P450P-2 (P4504A4) 379

6. Substrate Specificities of Rabbit Lung Cytochrome P450Isozymes 380

II. Rat Lung Microsomal Cytochrome P450 Isozymes 3811. Regulation and Characteristics of Rat Lung Cytochrome

P4502B1 3812. Regulation and Characteristics of Rat Lung Cytochromes

P4501A1 and P4501A2 382

Contents XXV

3. Regulation and Characteristics of Rat Lung CytochromeP4502A3 382

4. Regulation and Characteristics of Rat Lung CytochromeP4504B1 382

References 383

CHAPTER 25

Extrahepatic Microsomal Forms: Brain Cytochrome P450M. WARNER and J.-A. GUSTAFSSON 387

A. Introduction 387B. Quantitation and Identification of Specific Forms of Brain

Cytochrome P450 3871. Hepatic Microsomal Forms of Cytochrome P450 3872. Aromatase 3893. Estrogen 2-Hydroxylase 3904. 5a-Androstane-3p, 17p-Diol Hydroxylase 390

C. Induction of Brain Cytochrome P450 3911. Xenobiotic Induction 3912. Hormonal Induction 391

D. Mitochondrial Cytochromes P450 391E. Novel Physiological Functions 392F. Potential Toxicological Consequences 393G. Conclusions 394References 394

CHAPTER 26Cytochrome P450 in Primary and Permanent Liver Cell CulturesL.R. SCHWARZ and F.J. WIEBEL 399

A. Introduction 399B. Primary Hepatocyte Cultures 399

I. Decrease of Cytochrome P450 in Primary HepatocyteCultures 399

II. Measures for Maintaining Cytochrome P450 4001. Composition of Culture Media 4012. Cocultures and Extracellular Matrix 402

III. Induction in Primary Hepatocyte Cultures 403C. Permanent Cultures of Hepatic Cells 405

I. H4IIEC3 Rat Hepatoma Cells : . . . . 405II. HepG2 Human Hepatoma Cells 406

III. Prospects for Developing Metabolically Competent Cell Lines . 407References 407

XXVI Contents

CHAPTER 27

Cytochromes P450 in Genetically Engineered Cell Cultures: The GeneTechnological ApproachJ. DOEHMER and H. GREIM. With 1 Figure 415

A. The Gene Technological Approach to Cytochrome P450 415B. Genetically Engineered Cell Culture Systems 418

I. Escherichia coli 419II. Yeast 420

III. Mammalian Cells 421C. Advantages and Limitations 423References 425

Section IV: Modulation of Cytochrome P450 Levels

CHAPTER 28

Genetics: Animal and Human Cytochrome P450 PolymorphismsA.K. DALY and J.R. IDLE. With 1 Figure 433

A. Introduction 433B. Polymorphisms in Cytochrome P450 Genes 433C. Debrisoquine Polymorphism 434

I. Phenotypic Studies 434II. Biochemical and Molecular Biological Studies 434

III. Molecular Basis of the Poor Metaboliser Phenotype inHumans 435

IV. Molecular Basis of the Debrisoquine Metabolism Deficiency inthe DA Rat 437

V. Interethnic Variation in Debrisoquine Metabolism 438VI. Consequences of the Poor Metaboliser Phenotype 438

D. Mephenytoin Polymorphism 439I. Phenotypic Studies 439

II. Biochemical and Molecular Biological Studies 439E. Other Polymorphisms in Human Cytochrome P450 Genes 440F. Conclusion 442References 442

CHAPTER 29

Hormonal Regulation of Cytochrome P450 in Rat LiverR. KATO and Y. YAMAZOE. With 7 Figures 447

A. Introduction 447B. Gonadal Hormones 447

Contents XXVII

C. Growth Hormone 449D. Insulin 453E. Thyroid Hormone 454F. Species Differences and Organ-Specific Regulation 456References 456

CHAPTER 30Age- and Gender-Related Expression of Rat Liver Cytochrome P450D.E. RYAN and W. LEVIN. With 1 Figure 461

A. Introduction and Background 461B. Hormonal Regulation of Constitutive Cytochromes P450 461C. Cytochrome P450a and RLM2 (CYP2A Subfamily) 462D. Cytochromes P450b, P450c, P450d and P450e (CYP1A and CYP2B

Subfamilies) 464E. Cytochromes P450f, P450g, P450h, P450i and P450k (CYP2C

Subfamily) 465I. Cytochrome P450f (CYP2C7) 465

II. Cytochrome P450g (CYP2C13) 465III. Cytochrome P450h (CYP2C11) 466IV. Cytochrome P450i (CYP2C12) 467V. Cytochrome P450k (CYP2C6) 468

F. Cytochrome P450j (CYP2E Subfamily) 468G. Cytochromes P450p and P4501 (CYP3A Subfamily) 469H. Additional Sex-Specific Cytochromes P450 470References 470

CHAPTER 31Changes in Cytochrome P450 in SenescenceL.S. BlRNBAUM 477

A. Introduction 477B. Cytochrome P450 Content 478

I. Hepatic 478II. Extrahepatic 479

C. Specific Cytochrome P450 Isozymes 480I. Structural Studies 480

II. Enzymatic Studies 4811. Hepatic 4812. Extrahepatic 485

III. Molecular Studies • 485D. Induction '. 486E. Conclusions 488References 488

XXVIII Contents

CHAPTER 32Regulation of Cytochrome P450 ExpressionK. SOGAWA and Y. FUJII-KURIYAMA. With 1 Figure 493

A. Introduction 493B. Cytochrome P450 1 Family 493

I. CM-Acting Regulatory Elements 494II. trans-Acting Regulatory Factors 495

C. Cytochrome P450 2B Family 497D. Cytochrome P450 4A Family 498E. Conclusions 499References 499

CHAPTER 33Induction of Cytochromes P450 1 and P450 2 by XenobioticsE. BRESNICK. With 4 Figures 503

A. Introduction 503B. Historical Perspectives 504C. Induction of CYP1A1 505

I. Need for New Protein Synthesis 505II. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, Polycyclic Hydrocarbons

and Cytochrome P450 5061. TCDD Receptor or Ah Receptor 5072. Polycyclic Hydrocarbon-Binding Protein or 4S Protein 508

D. CYP1A1 and CYP1A2 Genes : 509I. Induction of the Cytochrome P450 1 Subfamily by Polycyclic

Hydrocarbons and Dioxins 510II. Regulation of the CYP1A1 Gene 512

1. Regulation by Polycyclic Hydrocarbons 514III. Further Notes on the Regulation of CYP1A2 514

E. Cytochrome P450 2 Family 515I. CYP2B1 and CYP2B2 515

1. Cytochrome P450 2B Genes and Transcription 5152. Phenobarbital Induction of Cytochrome P450 in Bacillus

megaterium 5163. Mechanism of Action of Phenobarbital as an Inducer of

CYP2B 516F. CYP2E1 517

I. Regulation of CYP2E1 517G. Conclusion 517References 518

Contents XXIX

Section V: Chemical Modification, Protein-Protein and Protein-LipidInteraction

CHAPTER 34

Protein-Protein InteractionsJ.B. SCHENKMAN. With 1 Figure 527

A. Introduction 527B. Microsomal Cytochrome P450 Monooxygenases 528

I. Cytochrome b5 Interactions 528II. NADPH-Cytochrome P450 Reductase Interactions 530

III. Cytochrome P450 Interactions 533C. Effects of Ionic Strength 534D. Role of the Hydrophobic Membrane-Binding Domain 537E. Mitochondrial P450 Monooxygenases 538

I. Adrenodoxin-Adrenodoxin Reductase Interactions 538II. Adrenodoxin-CYPllAl Interactions 539

F. Conclusions 541References 542

CHAPTER 35Chemical Probes of Cytochrome P450 StructureR. BERNHARDT. With 1 Figure 547

A. Introduction ; 547B. Identification and Localization of Functionally Important Regions

of Cytochrome P450 548I. Regions and Residues Forming the Substrate-Binding Site of

Cytochrome P450 5481. Modification of Cysteine Residues 5482. Modification of Histidine Residues 5493. Modification of Amino Groups 5494. Modification of Tyrosine Residues 550

II. Residues Involved in Cytochrome P450/Electron DonorInteractions 5501. Microsomal Cytochrome P450 Systems 5502. Mitochondrial Cytochrome P450 Systems 5523. Bacterial Cytochrome P450 Systems 553

C. Characterization of Structurally Important Regions of CytochromeP450 554

I. Accessibilities of Residues and Regions 554II. Location of Selected Amino Acids Residues with Respect to

Heme 555

XXX Contents

III. Location of Selected Residues with Respect to the MicrosomalMembrane 556

D. Concluding Remarks 556References 557

CHAPTER 36Posttranslational Modification of Cytochrome P450I. JANSSON 561

A. Introduction 561B. Phosphorylation of Cholesterol 7a-Hydroxylase (CYP7) 562C. Phosphorylation of Phenobarbital-Inducible (CYP2B4) and Other

Forms of Microsomal Cytochrome P450 with Broad SubstrateSpecificity 564

I. Phosphorylation of Cytochrome P450 564II. Possible Role of Phosphorylation 568

D. Phosphorylation of Mitochondrial Forms of Cytochrome P450 571E. Processing of Newly Synthesized Cytochrome P450 upon

Incorporation into Membranes 573F. Conclusions 574References 576

CHAPTER 37Lipid-Protein InteractionsJ. BLANCK and K. RUCKPAUL. With 1 Figure 581

A. Introduction 581B. Liver Microsomal Systems 581

I. Structural Aspects 582II. Dynamic Aspects 583

III. Functional Aspects 584IV. Protein-Lipid Interactions 586V. Physiological Implications 587

C. Adrenal Mitochondrial Systems 588I. Structural Aspects 588

II. Functional Aspects 589References 591

Section VI: Biosynthetic Forms of Cytochrome P450

CHAPTER 38Cholesterol 7a-Hydroxylase and 12a-HydroxylaseK. OKUDA, T. OGISHIMA, and M. NOSHIRO. With 1 Figure 601

Contents XXXI

A. Introduction 601B. Cholesterol 7a-Hydroxylase (CYP7) 601

I. Physiological Significance 601II. Purification and Properties 603

III. Cloning of cDNA and Gene Structure 603IV. Regulation 604

C. 12a-Hydroxylase 606I. Physiological Significance 606

II. Assay Method 606III. Purification and Properties 607IV. Regulation ' . . 607

D. Concluding Remarks 608References 608

CHAPTER 39

Tissue-Specific Regulation of Aromatase Cytochrome P450 (CYP19)ExpressionE.R. SIMPSON, M.S. MAHENDROO, G.D. MEANS, M.W. KILGORE,

C.J. CORBIN, and C.R. Mendelson. With 6 Figures 611

A. Introduction 611B. Comparison of the cDNA Insert Encoding Human Aromatase

Cytochrome P450 with That of Other Species 613C. Characterization of the Aromatase Cytochrome P450 Gene 615D. Regulation of Aromatase Gene Expression in Human Ovary 618E. Regulation of Aromatase Expression in Human Adipose 619F. Tissue-Specific Regulation of Human P450arom Expression Is

Achieved Using Alternative Promoters 620References 623

CHAPTER 40

Lanosterol 14a-Demethylase (Cytochrome P45014DM)Y. YOSHIDA. With 6 Figures 627

A. Introduction 627B. Molecular Properties 627C. Catalytic Features 631D. Structure of Substrate Necessary for Interaction with Cytochrome

P45014DM 633E. Substrate Specificities of Sterol Demethylase Cytochromes P450 of

Different Organisms 635F. Summary and Outlook 637References 637

XXXII Contents

CHAPTER 41

Steroid lip-Hydroxylase Isozymes (CYP11B1 and CYP11B2)P.C. WHITE, K.M. CURNOW, and L. PASCOE. With 2 Figures 641

A. Zonal Distribution of lip-Hydroxylase Activity 641B. Biochemistry 641C. Genetics 643D. Genetic Disorders of ll(3-Hydroxylase Isozymes 644

I. Steroid lip-Hydroxylase Deficiency 644II. Corticosterone Methyloxidase II Deficiency 645

III. Dexamethasone (or Glucocorticoid) SuppressibleHyperaldosteronism 646

IV. Types of Mutations Observed in the CYP11B Genes 646E. Summary 648References 648

CHAPTER 42

Cholesterol Side Chain Cleavage Cytochrome P450 (P450scc)L.E. VICKERY. With 3 Figures : 651

A. Introduction 651B. Mechanism of the Reaction 651C. Properties of Cytochrome P450scc 653D. Regulation of Activity 656E. Molecular Biology 657References _ 659

CHAPTER 43

Cytochrome P450cl7: Regulation of Gene Expression and EnzymeFunction at the Bifurcation in Steroid Hormone SynthesisW.N. KUHN-VELTEN 667

A. Introduction 667B. Localization and Developmental Changes 667C. Structural Characteristics and Expression of the CYP17 Gene 668D. Assumptions About Cytochrome P450cl7 Protein Structure 669E. Specificity of Ligand Binding 669F. Intrinsic and Extrinsic Regulation of Catalytic Properties 670G. Cytochrome P450cl7 as Target for Suppression of Steroid Hormone

Synthesis by Drugs , . 671H. The Problem of Cytochrome P450cl7 Downregulation: Role of

Substrate and Oxygen 672I. Pathophysiological Aspects 673References 673

Contents XXXIII

CHAPTER 4425-Hydroxyvitamin D-la-Hydroxylases: An Examination of Renal andExtrarenal SourcesB.W. HOLLIS and R.W. GRAY. With 5 Figures 677

A. Introduction 677I. Structure of Vitamin D 677

II. General Vitamin D Metabolism 677B. Renal 25-Hydroxyvitamin D-la-Hydroxylase 679

I. Metabolic Control 679II. Characterization, Isolation and Reconstitution 681

III. Molecular Mechanism of Regulation 683C. Extrarenal 25-Hydroxyvitamin D-la-Hydroxylase(s) 683

I. Localization in Tissues of Extrarenal Origin 683II. Biochemical Characteristics 685

III. Metabolic Control 686References 687

CHAPTER 45Steroid 21-HydroxylaseP.C. WHITE. With 1 Figure 693

A. Introduction 693B. Biochemistry 693C. Molecular Genetic Analysis 693D. Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency 695

I. Clinical Features 695II. Genetic Analysis 695

1. Salt-Wasting Form 6962. Simple Virilizing Form 6993. Nonclassic Form 699

E. Regulation 700F. Additional Progesterone 21-Hydroxylase activities 700G. Summary 701References 701

CHAPTER 46Sterol 26-HydroxyIaseK. WlKVALL , . . . . 705

A. Introduction 705B. Stereochemistry and Nomenclature of 26-Hydroxylation 705C. Functions of Sterol 26-Hydroxylase 706

XXXIV Contents

I. Bile Acid Biosynthesis 706II. Vitamin D3 Metabolism 707

III. Formation and Role of 26-Hydroxycholesterol in Metabolism . 707D. Purification and Characterization of Mitochondrial Cytochrome

P450 Active in 26-Hydroxylation 708I. Rabbit Mitochondrial Cytochrome P450 708

II. Rat Mitochondrial Cytochrome P450 711III. Pig Mitochondrial Cytochrome P450 712IV. Human Mitochondrial Cytochrome P450 and

Cerebrotendinous Xanthomatosis 713References 715

CHAPTER 47

Nitric Oxide Synthase (NOS)K.A. WHITE and M.A. MARLETTA. With 4 Figures 719

A. Introduction 719B. NOS Characteristics 719C. Characterization of the NOS as a Cytochrome P-450 721D. Mechanistic Implications ' 723E. Conclusion 725References 725

Subject Index r 729