Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme.
Based on the X-ray crystal structures of 4-(4-chlorophenyl)imidazole (4-CPI)- and bifonazole (BIF)-bound P450 2B4, eight active site mutants at six positions were created in an N-terminal modified construct termed 2B4dH and characterized for enzyme inhibition and catalysis. I363A showed a >4-fold decrease in differential inhibition by BIF and 4-CPI (IC(50,BIF)/IC(50,4-CPI)). F296A, T302A, I363A, V367A, and V477A showed a 2-fold decreased k(cat) for 7-ethoxy-4-trifluoromethylcoumarin O-deethylation, whereas V367A and V477F showed an altered K(m). T302A, V367L, and V477A showed >4-fold decrease in total testosterone hydroxylation, whereas I363A, V367A, and V477F showed altered stereo- and regioselectivity. Interestingly, I363A showed a 150-fold enhanced k(cat)/K(m) with testosterone, and yielded a new metabolite. Furthermore, testosterone docking into three-dimensional models of selected mutants based on the 4-CPI-bound structure suggested a re-positioning of residues 363 and 477 to yield products. In conclusion, our results suggest that the 4-CPI-bound 2B4dH/H226Y crystal structure is an appropriate model for predicting enzyme catalysis.
Hernandez
Cynthia E
CE
Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031, USA.
Kumar
Santosh
S
Liu
Hong
H
Halpert
James R
JR
D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D020134:Catalytic Domain / Q000235:genetics* / Q000502:physiology; D002855:Chromatography, Thin Layer; D003374:Coumarins / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D000072467:Cytochrome P450 Family 2; D004305:Dose-Response Relationship, Drug; D006900:Hydroxylation / Q000187:drug effects; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D013237:Stereoisomerism; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D000596:Amino Acids; D003374:Coumarins; D007093:Imidazoles; D008024:Ligands; C056033:7-ethoxy-4-trifluoromethylcoumarin; D013739:Testosterone; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); C036596:bifonazole