Abstract
The formation of a reversible adsorption complex between a dimer of N-acetyl-L-tyrosine [di-(N-acetyl-L-tyrosine), (NAT)2] and horseradish peroxidase (HRP) compound II (CII) was demonstrated using a kinetic approach. A specific KIIm value (0.58 mM) was deduced for this step from stopped-flow measurements. The dimerization of the dipeptide Gly-Tyr was analysed at the steady state and compared with (NAT)2 dimerization [(NAT)2-->(NAT)4]. A saturation of the enzyme was observed for both substrates within their range of solubility. In each case the rate of dimerization reflected the rate-limiting step of compound II reduction to the native HRP (E) (kappcat/Kappm approximately kII-->E). The kappcat values for (Gly-Tyr)2 and (NAT)4 formation were 254 s-1 and 3.6 s-1 respectively. The KappM value of Gly-Tyr was 24 mM. It was observed that the value (0.7 mM) for (NAT)2 was close both to its specific KIIm value for the second step of reduction (CII-->E) and to its thermodynamic dissociation constant (Kd=0.7 mM) with the resting form of the enzyme. As (NAT)2 was a tighter ligand but a poorer substrate than Gly-Tyr, a steady-state kinetic study was performed in the presence of both substrates. A kinetic model which includes an enzyme-substrate adsorption prior to each of the two steps of reduction was derived. This one agreed reasonably well with the experimental data.
Full Text
The Full Text of this article is available as a PDF (188.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adak S., Mazumder A., Banerjee R. K. Probing the active site residues in aromatic donor oxidation in horseradish peroxidase: involvement of an arginine and a tyrosine residue in aromatic donor binding. Biochem J. 1996 Mar 15;314(Pt 3):985–991. doi: 10.1042/bj3140985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Candeias L. P., Folkes L. K., Wardman P. Factors controlling the substrate specificity of peroxidases: kinetics and thermodynamics of the reaction of horseradish peroxidase compound I with phenols and indole-3-acetic acids. Biochemistry. 1997 Jun 10;36(23):7081–7085. doi: 10.1021/bi970384e. [DOI] [PubMed] [Google Scholar]
- Childs R. E., Bardsley W. G. The steady-state kinetics of peroxidase with 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as chromogen. Biochem J. 1975 Jan;145(1):93–103. doi: 10.1042/bj1450093. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Critchlow J. E., Dunford H. B. Studies on horseradish peroxidase. IX. Kinetics of the oxidation of p-cresol by compound II. J Biol Chem. 1972 Jun 25;247(12):3703–3713. [PubMed] [Google Scholar]
- Critchlow J. E., Dunford H. B. Studies on horseradish peroxidase. X. The mechanism of the oxidation of p-cresol, ferrocyanide, and iodide by compound II. J Biol Chem. 1972 Jun 25;247(12):3714–3725. [PubMed] [Google Scholar]
- Fornstedt B., Rosengren E., Carlsson A. Occurrence and distribution of 5-S-cysteinyl derivatives of dopamine, dopa and dopac in the brains of eight mammalian species. Neuropharmacology. 1986 Apr;25(4):451–454. doi: 10.1016/0028-3908(86)90242-x. [DOI] [PubMed] [Google Scholar]
- Graham D. G. Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones. Mol Pharmacol. 1978 Jul;14(4):633–643. [PubMed] [Google Scholar]
- Henriksen A., Schuller D. J., Meno K., Welinder K. G., Smith A. T., Gajhede M. Structural interactions between horseradish peroxidase C and the substrate benzhydroxamic acid determined by X-ray crystallography. Biochemistry. 1998 Jun 2;37(22):8054–8060. doi: 10.1021/bi980234j. [DOI] [PubMed] [Google Scholar]
- La Mar G. N., Hernández G., de Ropp J. S. H NMR investigation of the influence of interacting sites on the dynamics and thermodynamics of substrate and ligand binding to horseradish peroxidase. Biochemistry. 1992 Sep 29;31(38):9158–9168. doi: 10.1021/bi00153a007. [DOI] [PubMed] [Google Scholar]
- Malencik D. A., Sprouse J. F., Swanson C. A., Anderson S. R. Dityrosine: preparation, isolation, and analysis. Anal Biochem. 1996 Nov 15;242(2):202–213. doi: 10.1006/abio.1996.0454. [DOI] [PubMed] [Google Scholar]
- Marquez L. A., Dunford H. B. Kinetics of oxidation of tyrosine and dityrosine by myeloperoxidase compounds I and II. Implications for lipoprotein peroxidation studies. J Biol Chem. 1995 Dec 22;270(51):30434–30440. doi: 10.1074/jbc.270.51.30434. [DOI] [PubMed] [Google Scholar]
- Michon T., Chenu M., Kellershon N., Desmadril M., Guéguen J. Horseradish peroxidase oxidation of tyrosine-containing peptides and their subsequent polymerization: a kinetic study. Biochemistry. 1997 Jul 15;36(28):8504–8513. doi: 10.1021/bi963168z. [DOI] [PubMed] [Google Scholar]
- Ralston I. M., Dunford H. B. Horseradish peroxidase. XLII. Oxidations of L-tyrosine and 3,5-diiodo-L-tyrosine by compound II. Can J Biochem. 1980 Nov;58(11):1270–1276. doi: 10.1139/o80-170. [DOI] [PubMed] [Google Scholar]
- Ralston I., Dunford H. B. Horseradish peroxidase. XXXII. pH dependence of the oxidation of L-(-)-tyrosine by compound I. Can J Biochem. 1978 Dec;56(12):1115–1119. doi: 10.1139/o78-175. [DOI] [PubMed] [Google Scholar]
- Ryu K., Dordick J. S. How do organic solvents affect peroxidase structure and function? Biochemistry. 1992 Mar 10;31(9):2588–2598. doi: 10.1021/bi00124a020. [DOI] [PubMed] [Google Scholar]
- Savenkova M. L., Mueller D. M., Heinecke J. W. Tyrosyl radical generated by myeloperoxidase is a physiological catalyst for the initiation of lipid peroxidation in low density lipoprotein. J Biol Chem. 1994 Aug 12;269(32):20394–20400. [PubMed] [Google Scholar]
- Schejter A., Lanir A., Epstein N. Binding of hydrogen donors to horseradish peroxidase: a spectroscopic study. Arch Biochem Biophys. 1976 May;174(1):36–44. doi: 10.1016/0003-9861(76)90321-0. [DOI] [PubMed] [Google Scholar]
- Schonbaum G. R. New complexes of peroxidases with hydroxamic acids, hydrazides, and amides. J Biol Chem. 1973 Jan 25;248(2):502–511. [PubMed] [Google Scholar]