Abstract
Binding of aromatic substrate molecules to myeloperoxidase has been investigated by EPR spectroscopy and model building. Binding of aromatic substrate molecules, such as phenol, p-cresol, resorcinol, and 4-amino salicylate, replaces the original rhombic high spin EPR spectrum of the ferric enzyme (g = 6.74, 5.18, and 1.97) by another high spin signal (g = 7.04, 4.87, and 1.93) indicating that these substrate molecules bind near the heme center of the enzyme. Salicylhydroxamic acid and benzohydroxamic acid complexes of myeloperoxidase showed EPR spectra composed of high spin (g = 6.99, 4.93, and 1.95) and low spin (2.66, 2.22, and 1.81) signals. The hydroxamic side chains of these two substrates seem to interact with the heme iron. Model building based on the three-dimensional structure of the enzyme (Zeng, J., and Fenna, R. E. (1992) J. Mol. Biol. 226, 185-207) revealed the presence of a hydrophobic pocket at the entrance of the distal heme cavity where the aromatic ring of these substrates can bind. Moreover, the six-membered ring portion of salicylhydroxamic acid and benzohydroxamic acid could bind to this hydrophobic pocket with the hydroxamic side chain placed between the imidazole of the distal His and the heme iron. The EPR results on lactoperoxidase and intestinal peroxidase also suggest the presence of an aromatic substrate binding site similar to that of myeloperoxidase.
Highlights
Binding of aromatic substrate moleculetso myeloper- oxidation reactions carried out by mammalian peroxidase enoxidase has been investigatebdy EPR spectroscopy and zymes are in principle the same as those catalyzed by plant modelbuilding.Binding of aromaticsubstratemol- peroxidasessuch as HRP(MorrisonandSchonbaum,1976)
Ecules, such as phenol,p-cresol,resorcinol, and 4-amino The mechanism of formation of the active compound I upon salicylate,replaces the original rhombic high spinEPR reaction with peroxides has been established by Poulos and spectrum of the ferric enzyme(g= 6.74,5.18, and 1.9b7y) Kraut through the x-ray structure determination of yeast cyanother high spin signal (g = 7.04, 4.87, and 1.93) indi- tochrome c peroxidase
Benzohydroxamicacidcomplexes of myeloperoxidase The presence of these residues in the activesite of MPO (Bolshowed EPR spectra composed of high spin (g = 6.99, scher and Wever; 1984a; Ikeda-Saito, 1985) with stereochem
Summary
(Received for publication, August 26, 1993, and in revised form, January 10, 1994). From the Wepartment of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, the §Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560 Japan, the llDepartment of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101-6129, and the IUaboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland20892. Salicylhydroxamic acid and His and distal His and Arg residues present in the active site. The EPR results on lactoperoxidase andintestinal per- strates (1989)suggested that phenolic substrates zymes found in mammalian body fluids and tissues They un- bind about 10A away from the heme iron in LPO, awnde dergo a 2 eq oxidation by peroxides, and carry out concludedfrombindingstudies that the aromatic substrate. 2-electron oxidationsof halides and 1-electron oxidatioonf s moleculesbindnear the distalhemepocketinmammalian aromatic molecules, such as phenol and quinone derivatives peroxidases, including MPO, LPO, and IPO The abbreviations used are: MPO, myeloperoxidase;4AS; 4-aminosalicylate; SHA, salicylhydroxamic acid; BHA, benzohydroxamic acid; LPO, lactoperoxidase; IPO,intestinal peroxidase; HRP,horseradish the entrance to the distal heme pocket.
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