Evidence for a radical mechanism in peroxidase-catalyzed coupling. II. Single turnover experiments with horseradish peroxidase.
Single turnover experiments were performed with horseradish peroxidase (HRP) to study the mechanism of peroxidase-catalyzed coupling and its stimulation by low concentrations of free diiodotyrosine (DIT). HRP was used because, unlike thyroid peroxidase (TPO) and lactoperoxidase (LPO), the spectral properties of compounds I and II are readily distinguishable. This made it possible to correlate the kinetics and stoichiometry of T4 + T3 formation with spectral data. Incubation of 2 microM preformed HRP-I with 2 microM [125I]Tg (thyroglobulin of low hormone content, high iodotyrosine content) in the presence of 1 microM free DIT yielded about 0.8 residue T4 and 0.2 residue T3 per molecule of Tg. This represents the theoretical maximum for iodothyronine formation, indicating remarkably efficient use of the oxidizing equivalents in HRP-I for coupling. The time course for formation of T4 + T3 was biphasic. During a rapid initial phase (about 1 min), HRP-I was completely converted to HRP-II, coincident with the formation of about 0.65 residues of T4 + T3. During the second slower phase, lasting 10-15 min, HRP-II was completely reduced to the native enzyme, with formation of the remaining T4 + T3. In the absence of DIT, the coupling yield was reduced to 0.5-0.6 residue T4 + T3 per molecule Tg, and the reaction, although considerably slower, was still biphasic. The rapid phase again corresponded to the conversion of HRP-I to HRP-II, and the slower phase to the conversion of HRP-II to native enzyme. To gain insight into the mechanism of the stimulatory effect of free DIT on coupling, we studied the reaction of DIT with HRP-I and HRP-II. Free DIT reacted with both HRP-I and HRP-II in one-electron transfer reactions, and the time course for these reductions resembled those observed with DIT + Tg. These observations suggest that in DIT-stimulated coupling, free DIT radicals act as a shuttle for transferring oxidizing equivalents from the peroxidase intermediates to the DIT residues in Tg. The remarkable efficiency of the HRP-I-mediated coupling reaction implies that (i) only hormonogenic residues in Tg are oxidized and (ii) oxidation of two hormonogenic residues occurs within the same molecule of Tg. A scheme which attempts to explain both kinetic and stoichiometric features of the coupling reaction observed in this study is proposed. This scheme is based on a radical mechanism, consistent with the conclusions reached in the companion paper.
Doerge
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DR
National Center for Toxicological Research, Jefferson, Arkansas 72079-9502.
Taurog
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A
Dorris
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ML
D000693:Anaerobiosis; D002374:Catalase / Q000378:metabolism; D004105:Diiodotyrosine / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005609:Free Radicals; D006735:Horseradish Peroxidase / Q000378:metabolism*; D008956:Models, Chemical; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013954:Thyroglobulin / Q000378:metabolism; D013974:Thyroxine / Q000096:biosynthesis*; D014284:Triiodothyronine / Q000096:biosynthesis*
D005609:Free Radicals; D014284:Triiodothyronine; D004105:Diiodotyrosine; D013954:Thyroglobulin; D006735:Horseradish Peroxidase; D002374:Catalase; D013974:Thyroxine