Mechanism-based inactivation of human cytochrome P450 1A2 by furafylline: detection of a 1:1 adduct to protein and evidence for the formation of a novel imidazomethide intermediate

Biochemistry. 1998 May 19;37(20):7407-19. doi: 10.1021/bi973011m.


The rapid loss of human CYP1A2 (cytochrome P450 1A2) activity caused by the 8-methylxanthine furafylline is investigated with the aim of determining whether a stable covalent adduct of the xanthine to the enzyme could be identified. Metabolic studies employing expressed CYP1A2 with radiolabeled furafylline and a close analogue, cyclohexylline, where the furan ring is replaced with cyclohexane, indicate that these xanthines are bound in a 1:1 ratio to CYP1A2 protein. This result, combined with earlier kinetic studies, verifies that these compounds are mechanism-based inhibitors of the enzyme. The 8'-methyl carbinols are the only metabolites formed by CYP1A2, and substantial (70-80%) incorporation of oxygen from the medium into the carbinols is observed. Carbinol formation is further characterized by high intramolecular isotope effects (kH/kD > 9) and low intermolecular isotope effects (DV/K < 2). Overall partition ratios are low (5.0 and 7.6, respectively), confirming our previous conclusion that furafylline is an efficient inactivator. By contrast, the N7-methyl-8-methylxanthines are good substrates for CYP1A2 but are not themselves inactivating agents. In addition to other metabolic products, the 8'-methyl carbinols of these N7-methyl-8-methylxanthines are formed in substantial amounts with equally high intramolecular isotope effects; however, the carbinol oxygen is derived exclusively from molecular oxygen. We conclude that oxidation of the 8-methyl group of furafylline and cyclohexylline, but not their N7-methyl analogues, by CYP1A2 promotes a major fraction of the inactivating xanthines to a two electron oxidized intermediate which either terminates enzyme activity by reaction with an active site amino acid or is decomposed by reaction with the medium to give carbinol.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Line, Transformed
  • Cytochrome P-450 CYP1A2 / metabolism
  • Cytochrome P-450 CYP1A2 Inhibitors*
  • Deuterium
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation / drug effects
  • Humans
  • Imidazoles / metabolism*
  • Microsomes, Liver / enzymology
  • Oxygen / metabolism
  • Theophylline / analogs & derivatives*
  • Theophylline / metabolism
  • Theophylline / pharmacology
  • Xanthines / pharmacology


  • Cytochrome P-450 CYP1A2 Inhibitors
  • Imidazoles
  • Xanthines
  • methylxanthine
  • Deuterium
  • Theophylline
  • furafylline
  • Cytochrome P-450 CYP1A2
  • Oxygen