Dual role of human cytochrome P450 3A4 residue Phe-304 in substrate specificity and cooperativity.
The structural basis of cooperativity of progesterone hydroxylation catalyzed by human cytochrome P450 3A4 has been investigated. A recent study suggested that substitution of larger side chains at positions Leu-211 and Asp-214 partially mimics the action of effector by reducing the size of the active site. Based on predictions from molecular modeling that Phe-304 in the highly conserved I helix is involved in both effector and substrate binding, a tryptophan residue was substituted at this position. The purified F304W mutant displayed hyperbolic progesterone hydroxylase kinetics, indicating a lack of homotropic cooperativity. However, the mutant remained responsive to stimulation by alpha-naphthoflavone, exhibiting a 2-fold decrease in the K(m) value for progesterone 6beta-hydroxylation in the presence of 25 microM effector. Combining substitutions to yield the triple mutant L211F/D214E/F304W maintained the V(max) and decreased the K(m) for progesterone 6beta-hydroxylation, minimized stimulation by alpha-naphthoflavone, and decreased the rate of alpha-naphthoflavone oxidation to one-eighth of the wild type. Interestingly, the DeltaA(max) for spectral binding of alpha-naphthoflavone was unaltered in L211F/D214E/F304W. Overall, the results suggest that progesterone and alpha-naphthoflavone are oxidized at separate locations within the P450 3A4 binding pocket, although both substrates appear to have equal access to the reactive oxygen.
Domanski
T L
TL
University of Texas Medical Branch, Department of Pharmacology and Toxicology, Galveston, Texas 77555-1031, USA. tadomans@utmb.edu
He
Y A
YA
Harlow
G R
GR
Halpert
J R
JR
D001189:Aryl Hydrocarbon Hydroxylases; D001571:Benzoflavones / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics / Q000502:physiology; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013250:Steroid Hydroxylases / Q000378:metabolism; D013379:Substrate Specificity
D001571:Benzoflavones; D010649:Phenylalanine; D011374:Progesterone; C011512:alpha-naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C022657:steroid hormone 6-beta-hydroxylase; C022724:testosterone 7-alpha-hydroxylase, hamster