Covalent alteration of the CYP3A4 active site: evidence for multiple substrate binding domains

Arch Biochem Biophys. 2001 Jul 1;391(1):49-55. doi: 10.1006/abbi.2001.2401.


The inhibition of CYP3A4-mediated oxidation of triazolam and testosterone was assessed in the presence of a selection of known CYP3A4 substrates and inhibitors. Under experimental conditions where the Michaelis-Menten model predicts substrate-independent inhibition ([S] = K(m)), results yielded substrate-dependent inhibition. Moreover, when the same experimental design was extended to a group of structurally similar flavonoids it was observed that flavanone, flavone, 3-hydroxyflavone, and 6-hydroxyflavone (10 microM) activated triazolam metabolism, but inhibited testosterone hydroxylation. In additional studies, residual CYP3A4 activity toward testosterone and triazolam hydroxylation was measured after pretreatment with the CYP3A4 mechanism based inhibitor, midazolam. After midazolam preincubation, CYP3A4 6 beta-hydroxylase activity was reduced by 47% while, in contrast, triazolam hydroxylation was reduced by 75%. These results provide physical evidence, which supports the hypothesis that the active site of CYP3A4 contains spatially distinct substrate-binding domains within the enzyme active site.

MeSH terms

  • Binding Sites
  • Binding, Competitive
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System / chemistry
  • Cytochrome P-450 Enzyme System / metabolism*
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Kinetics
  • Microsomes, Liver / enzymology
  • Microsomes, Liver / metabolism
  • Midazolam / pharmacology
  • Mixed Function Oxygenases / antagonists & inhibitors
  • Mixed Function Oxygenases / chemistry
  • Mixed Function Oxygenases / metabolism*
  • Protein Structure, Tertiary
  • Substrate Specificity
  • Testosterone / metabolism*
  • Triazolam / metabolism*


  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Triazolam
  • Testosterone
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • CYP3A protein, human
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human
  • Midazolam