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.
Highlights
The reaction of horseradish peroxidase with alkyl- Compound I, aferryl porphyrin radical cation that can be hydrazines results in 6-meso-alkylation of the pros- representedas(porphyrint)Fe” = 0 [1, 2].Cytothetic heme group and enzyme inactivation
Little is known about the structural formation of the isoporphyrin intermediate at 25 “C and electronic factorsthatdeterminewhetherthe second are, respectively, 11.6 and 12.5 min for methylhydra- oxidation equivalent is located on the porphyrin or the prozine (2.0mM), 8.7 and 7.2min for ethylhydrazine(1.0 tein
Spectra of the Isoporphyrin Intermediates-The spectra of nm rnin the isoporphyrin intermediates generated in the reactions of Methyl (2.0 mM)
Summary
Spectra of the Isoporphyrin Intermediates-The spectra of nm rnin the isoporphyrin intermediates generated in the reactions of Methyl (2.0 mM). 8310.102.151.6 horseradish peroxidase with methyl-, ethyl-,n-bu45ty0l-,an7d.2 8.7Ethy8l 4(01.0 mM). 4.2 hibit a near-infrared absorbance band and an attenuated and Phenylethyl (50 pM) 825 0.5 0.3 0.4 slightly red-shifted Soret maximum (Fig. 2). *Experimentswere carried out at 25 "C with 10 p M horseradish peroxidase except with phenylethylhydrazine, for which a 1p M horseradish peroxidase concentration was used. Soret band sharpens and shifts from 416 to 421 nm but the long wavelength band remains unaltered (Fig. 2). Reduction of the isoporphyrin with dithionite in the presence of carbon monoxide sharpens the Soretbandandshiftsit to longer wavelengths and attenuates the long wavelength band (Fig. 2)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
