Heme Modification Contributes to the Mechanism-Based Inactivation of Human Cytochrome P450 2J2 by Two Terminal...

Area of Science:

• Biochemistry
• Pharmacology
• Enzyme kinetics

Background:

• Cytochrome P450 enzymes, including CYP2J2, are crucial for drug metabolism.
• Understanding enzyme inactivation mechanisms is vital for predicting drug-drug interactions and drug efficacy.
• Terminal acetylenic compounds are known to be mechanism-based inhibitors of P450 enzymes.

Purpose of the Study:

• To investigate the mechanism-based inactivation of human CYP2J2 by three terminal acetylenic compounds: N-(methylsulfonyl)-6-(2-propargyloxyphenyl)hexanamide (MS), 17-octadecynoic acid (OD), and danazol (DZ).
• To elucidate the specific molecular interactions leading to CYP2J2 inactivation by these compounds.

Main Methods:

• Assay of hydroxyebastine (OHEB) carboxylation activity in a reconstituted CYP2J2 system.
• Kinetic analysis of mechanism-based inactivation, including K_I and k_inact determination.
• Spectroscopic analysis (heme spectrum, reduced CO difference spectrum) and LC-MS for adduct identification.

Main Results:

• MS and OD caused time- and concentration-dependent inactivation of CYP2J2, requiring NADPH and leading to heme modification.
• Kinetic parameters (K_I, k_inact) and partition ratios were determined for MS and OD.
• Danazol (DZ) caused inactivation via a different mechanism, indicated by a Type II binding spectrum and lower IC50, without apparent heme modification.

Conclusions:

• Heme modification by MS and OD is responsible for the mechanism-based inactivation of CYP2J2.
• The ethynyl moiety of MS and OD likely interacts with the heme iron.
• Danazol's inactivation mechanism involves preferential orientation of its isoxazole ring toward the heme iron.