Abstract
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 α-naphthoflavone, exhibiting a 2-fold decrease in theKm value for progesterone 6β-hydroxylation in the presence of 25 μM effector. Combining substitutions to yield the triple mutant L211F/D214E/F304W maintained theVmax and decreased theKm for progesterone 6β-hydroxylation, minimized stimulation by α-naphthoflavone, and decreased the rate of α-naphthoflavone oxidation to one-eighth of the wild type. Interestingly, the ΔAmax for spectral binding of α-naphthoflavone was unaltered in L211F/D214E/F304W. Overall, the results suggest that progesterone and α-naphthoflavone are oxidized at separate locations within the P450 3A4 binding pocket, although both substrates appear to have equal access to the reactive oxygen.
Footnotes
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Send reprint requests to: Dr. Tammy L. Domanski, Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031. E-mail: tadomans{at}utmb.edu
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↵1 This study was supported by National Research Award GM19058, National Institutes of Health Grant GM54995, and Center Grant ES06676.
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Received for publication
- Abbreviations:
- P450
- cytochrome P450
- ANF
- α-naphthoflavone
- PCR
- polymerase chain reaction
- DOPC
- dioloeoylphosphatidylcholine
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- E
- enzyme
- S
- substrate
- WT
- wild type
- Accepted December 23, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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