Propyl Gallate Inhibition of Iodide and Tetramethylbenzidine Oxidation Catalyzed by Human Thyroid Peroxidase

E. E. Grintsevich¹, V. V. Senchuk², A. V. Puchkaev¹, and D. I. Metelitza^1*

¹Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Kuprevicha 5/2, Minsk, 220141 Belarus; fax: (10-375-17) 63-7274; E-mail: enzyme@ns.iboch.ac.by

²School of Biology, Belorussian State University, ul. Kurchatova 5, Minsk, 220106 Belarus; fax: (10-375-17) 77-5535; E-mail: biochem@bio.bsu.unibel.by

^* To whom correspondence should be addressed.

Received July 7, 1999; Revision received October 5, 1999

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The kinetic characteristics (k_cat, K_m, and their ratio) for oxidation of iodide (I^-) at 25°C in 0.2 M acetate buffer, pH 5.2, and tetramethylbenzidine (TMB) at 20°C in 0.05 M phosphate buffer, pH 6.0, with 10% DMF catalyzed by human thyroid peroxidase (HTP) and horseradish peroxidase (HRP) were determined. The catalytic activity of HRP in I^- oxidation was about 20-fold higher than that of HTP. The k_cat/K_m ratio reflecting HTP efficiency was 35-fold higher in TMB oxidation than that in I^- oxidation. Propyl gallate (PG) effectively inhibited all four peroxidase processes and its effects were characterized in terms of inhibition constants K_i and the inhibitor stoichiometric coefficient f. For both peroxidases, inhibition of I^- oxidation by PG was characterized by mixed-type inhibition; K_i for HTP was 0.93 µM at 25°C. However, in the case of TMB oxidation the mixed-type inhibition by PG was observed only with HTP (K_i = 3.9 µM at 20°C), whereas for HRP it acted as a competitive inhibitor (K_i = 42 µM at 20°C). A general scheme of inhibition of iodide peroxidation containing both enzymatic and non-enzymatic stages is proposed and discussed.

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KEY WORDS: human thyroid peroxidase, horseradish peroxidase, iodide-ion, tetrametylbenzidine, peroxidase oxidation, propyl gallate, inhibition, inhibition constants, co-oxidation, peroxidase catalysis

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