title: Stabilized isoporphyrin intermediates in the inactivation of horseradish peroxidase by alkylhydrazines.
abstract: The reaction of horseradish peroxidase with alkylhydrazines results in delta-meso-alkylation of the prosthetic heme group and enzyme inactivation (Ator, M. A., David, S. K., and Ortiz de Montellano, P. R. (1987) J. Biol. Chem. 262, 14954-14960). As reported here, enzyme inactivation is associated with the accumulation of intermediates that absorb at approximately 835 nm. The properties of these intermediates, including their collapse to give meso-alkylhemes, identify them as isoporphyrins. The t1/2 values for inactivation and formation of the isoporphyrin intermediate at 25 degrees C are, respectively, 11.6 and 12.5 min for methylhydrazine (2.0 mM), 8.7 and 7.2 min for ethylhydrazine (1.0 mM), and 30 and 25 s for phenylethylhydrazine (50 microM). The isoporphyrin intermediates are surprisingly long-lived, with half-lives (35 degrees C, pH 7.0) of 9, 28, 96, and 450 min for, respectively, the phenylethyl, methyl, n-butyl, and ethyl analogues. pH studies show that protonation of a group with pKa = 5.0-6.5 accelerates isoporphyrin decay and decreases steady state isoporphyrin accumulation. Horseradish peroxidase reconstituted with delta-meso-methylheme, unlike horseradish peroxidase with a heme that has a larger meso-substituent, is catalytically active but is more sensitive to H2O2-mediated degradation of the prosthetic group than is the native enzyme. The delta-meso-methylheme prosthetic group is converted in the reaction with H2O2 to a biliverdin-like product. The results implicate highly stabilized isoporphyrin intermediates in the inactivation of horseradish peroxidase by alkylhydrazines and indicate that inactivation by the meso-alkyl groups is due to steric interference with electron delivery to the heme edge rather than to intrinsic electronic consequences of meso-alkylation. The structural features that stabilize the cationic isoporphyrins may also be involved in stabilization of the Compound I porphyrin radical cation.
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
authors.forename: M A
authors.initials: MA
authors.lastname: Ator
authors.forename: S K
authors.initials: SK
authors.lastname: David
authors.forename: P R
authors.initials: PR
authors.lastname: Ortiz de Montellano
mesh_terms: D000478:Alkylation; D002384:Catalysis; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006418:Heme; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006834:Hydrazines / Q000494:pharmacology*; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D010544:Peroxidases / Q000378:metabolism*; D011166:Porphyrins; D011190:Potassium Cyanide; D013053:Spectrophotometry
chemical_list: D006834:Hydrazines; D011166:Porphyrins; D006418:Heme; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010544:Peroxidases; D011190:Potassium Cyanide