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Amino acid 305 determines catalytic center accessibility in CYP3A4
S M Fowler1, R J Riley, M P Pritchard
1Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee DD1 9SY, U.K. stephen.fowler@astrazeneca.com
Biochemistry
|April 12, 2000
Summary
Site-directed mutagenesis of cytochrome P450 3A4 (CYP3A4) revealed that replacing alanine 305 with phenylalanine (A305F) significantly alters enzyme activity and inhibitor binding. The A305F mutation reduces metabolism of several substrates and decreases binding affinity for azole inhibitors.
Area of Science:
- Biochemistry and Molecular Biology
- Enzymology
- Drug Metabolism and Pharmacokinetics
Background:
- Cytochrome P450 3A4 (CYP3A4) is a critical enzyme in drug metabolism.
- Understanding the structure-activity relationship of CYP3A4 is essential for predicting drug-drug interactions and optimizing drug design.
- Specific amino acid residues in the active site can significantly influence substrate binding and inhibitor interactions.
Purpose of the Study:
- To investigate the functional impact of alanine 305 mutations (to phenylalanine and serine) in the CYP3A4 active site.
- To determine how these mutations affect the enzyme kinetics of various substrates.
- To assess the influence of these mutations on the binding affinity of CYP3A4 inhibitors.
Main Methods:
- Site-directed mutagenesis was employed to create CYP3A4 variants A305F and A305S.
- Enzyme kinetics (S50 and Vmax) were determined for the metabolism of diazepam, erythromycin, nifedipine, and testosterone using wild-type and mutant enzymes.
- Inhibition constants (Ki) were measured for selective and broad-specificity azole inhibitors (e.g., ketoconazole, clotrimazole, econazole, miconazole).
Main Results:
- The A305F mutation abolished diazepam oxidation and significantly reduced Vmax and/or increased S50 for erythromycin, testosterone, and nifedipine metabolism.
- A305F substantially increased the Ki values for ketoconazole and other azole inhibitors, indicating reduced binding affinity.
- The A305S mutation enhanced testosterone and erythromycin metabolism but reduced nifedipine metabolism, with no significant change in ketoconazole Ki values.
Conclusions:
- Mutation of alanine 305 to phenylalanine in CYP3A4 increases steric hindrance in the active site, decreasing substrate metabolism and azole inhibitor binding.
- The A305S mutation has differential effects on substrate metabolism, suggesting a complex role for residue 305.
- These findings provide insights into CYP3A4 active site topology and its implications for drug-substrate and drug-inhibitor interactions.
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