Catalytic properties of horseradish peroxidase reconstituted with the 8-(hydroxymethyl)- and 8-formylheme derivatives.
Recent studies suggest that 8-(thiomethyl)- and 8-formylheme modifications may be present in, respectively, lactoperoxidase and myeloperoxidase. To examine whether these heme modifications contribute to the unusual catalytic properties of the mammalian peroxidases, we have reconstituted apo-horseradish peroxidase (HRP) with 8-(hydroxymethyl)heme (8HM-HRP) and 8-formylheme (8F-HRP) and have characterized the reconstituted enzymes. Native HRP and 8HM-HRP have identical spectra in the ferric, compound I, and compound II states. In contrast, the Soret band of 8F-HRP is at 417 rather than 402 nm and that of its compound II species is at 436 rather than 416 nm. Compound I was observed as a transient species with 8F-HRP. The rate of formation of compound I was the same for native and 8HM-HRP, but the pseudo-first-order constant for decay of compound I was 0.021 s-1 for 8HM-HRP and 0.010 s-1 for native HRP. The rates of oxidation of guaiacol, iodide, and thioanisole are the same for native HRP and 8HM-HRP but are significantly slower for 8F-HRP. The stereospecificity of thioanisole oxidation is the same for native and 8HM-HRP, but differs for 8F-HRP. For guaiacol, which was studied in detail, Km = 2.3 mM and kcat = 33 s-1 for 8F-HRP versus Km = 1.8 mM and kcat = 104 s-1 for native HRP. 8HM-HRP oxidizes ethylhydrazine and azide to the ethyl and azidyl radicals, respectively, and is simultaneously inactivated. 8F-HRP is also slowly inactivated by ethylhydrazine and azide.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
R Z
RZ
Harris
P A
PA
Liddell
K M
KM
Smith
P R
PR
Ortiz de Montellano
D000840:Anisoles / Q000378:metabolism; D001386:Azides / Q000494:pharmacology; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D006139:Guaiacol / Q000378:metabolism; D006418:Heme / Q000031:analogs & derivatives*; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D006834:Hydrazines / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D006900:Hydroxylation; D007454:Iodides / Q000378:metabolism; D007700:Kinetics; D008745:Methylation; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013329:Structure-Activity Relationship
D000840:Anisoles; D001386:Azides; D006834:Hydrazines; D007454:Iodides; D006418:Heme; D006139:Guaiacol; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase