pH-dependent one- and two-electron oxidation of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine catalyzed by horseradish peroxidase.
The porphyrinogenic agent 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) is known to inactivate hepatic cytochrome P450 (P450) enzymes 2C11, 2C6, and 3A1 [Correia et al. (1987) Arch. Biochem. Biophys. 258, 436-451] by different mechanisms. The inactivation of P450 2C11 and 2C6 appears to be due to the ethylation of the heme in the active sites of the enzymes [Augusto et al. (1982) J. Biol. Chem. 257, 11288-11295], whereas the inactivation of P450 3A1 appears to involve the covalent binding of the heme to the apoprotein [Correia et al. (1987)]. Moreover, we have found that DDEP inactivates horseradish peroxidase (HRP) pretreated with hydrogen peroxide. In this system, DDEP was oxidized predominately to 3,5-dicarbethoxy-2,6-dimethyl-4-ethylpyridine (EDP) under weakly acidic conditions and predominately to 3,5-dicarbethoxy-2,6-dimethylpyridine (DP) under basic conditions. The loss of heme and the formation of altered heme products were also pH-dependent and were correlated with the formation of DP and the inactivation of HRP. Thus the inactivation of HRP appears to depend on the formation of an ethyl radical, which presumably reacts with the heme in the active site of the enzyme. Similar product ratios were obtained for the oxidation of DDEP by K3Fe(CN)6, indicating that product ratios of DP over EDP are mainly determined by the pH of buffer. These results, in addition to semiemperical calculations (AM1) for the oxidation of DDEP in the gas phase, are consistent with the idea that the inhibitor undergoes a single-electron oxidation to form the DDEP radical cation, the fate of which depends on the environment of the active site of the enzyme. The proposed formation of a radical cation by the abstraction of an electron from nitrogen is consistent with the finding of low intramolecular isotope effects of the metabolism of 3,5-dicarbethoxy-2,6-dimethyl-[4-2H,4-1H]-1,4-dihydropyridine by P450 2C11 and 3A4. Under basic or aprotic conditions, the radical dissociates to form DP and the ethyl radical, which reacts with the heme, thereby inactivating the enzyme. Under acidic or polar conditions, the radical undergoes an additional one-electron oxidation to form EDP.(ABSTRACT TRUNCATED AT 400 WORDS)
Sugiyama
K
K
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Correia
M A
MA
Thummel
K E
KE
Nagata
K
K
Darbyshire
J F
JF
Osawa
Y
Y
Gillette
J R
JR
D002384:Catalysis; D065607:Cytochrome P-450 Enzyme Inhibitors; D003997:Dicarbethoxydihydrocollidine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D004583:Electrons; D005295:Ferrocyanides / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D011165:Porphyrinogens / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology
D065607:Cytochrome P-450 Enzyme Inhibitors; D005295:Ferrocyanides; D011165:Porphyrinogens; C030826:3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine; D006418:Heme; D003997:Dicarbethoxydihydrocollidine; D006735:Horseradish Peroxidase