J. Biochem. 108, 804-810 (1990)
Iodothyronine-Induced Catalatic Activity of Thyroid Peroxidase Masao Nakamura,* Isao Yamazaki,*1' and Sachiya Ohtaki** 'Biophysics Division, Research Institute of Applied Electricity, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060; and *' Central Laboratory of Clinical Investigation, Miyazaki Medical College Hospital, Kiyotake, Miyazaki 889-16
Iodothyronines induced catalatic (H202-decomposing) activity of thyroid peroxidase and lactoperoxidase, the effect increasing in the order of thyroxine (T4)>triiodothyronine (T3) > diiodothyronine (T2). The iodothyronines served as electron donors in the peroxidase reactions, and during the reactions the catalytic intermediate of thyroid peroxidase was confirmed to be Compound II for T4 and Compound I for T3 and T2 and from the Soret absorption spectra obtained by stopped-flow measurements. Rate constants for the reactions between T4 and Compound II, T3 and Compound I, and T2 and Compound I were estimated at 1.9X 10s,1.3X 106, and 7.1 X10s M 1 - s 1 , respectively. Unlike the case of thyroid peroxidase, the catalytic intermediate of lactoperoxidase observed during the oxidation of iodothyronines was invariably Compound II. From these and other data it was concluded that thyroid peroxidase catalyzed one-electron oxidation of T4 and two-electron oxidations of T2 and T3 while lactoperoxidase catalyzed exclusively one-electron oxidation of the iodothyronines. Iodide was released during the enzymatic oxidation of iodothyronines, irrespective of the mechanism of one-electron and two-electron oxidations. The amount of released iodide increased in the order of T4 >T3 >T2. The iodothyronines-induced catalatic activity of these peroxidases was ascribable to the release of iodide, but it was also found that the iodide-enhanced catalatic activity was stimulated by iodothyronines. In this case the effect of iodothyronines was greater in the order of T2 >T3 >T4, which was consistent with the order of iodothyronine activation for the iodinium cation transfer from enzyme to acceptor.
It has been demonstrated that peroxidases have more or less catalatic (H2O2-decomposing) activity (1-6), which increases in the presence of halides (1, 2, 6, 7). In particular, thyroid peroxidase and lactoperoxidase exhibit a strong iodide-dependent catalatic activity (2, 7). The two-electron oxidation of iodide has been confirmed in the reactions of horseradish peroxidase (8, 9), lactoperoxidase, and thyroid peroxidase (10), resulting in the formation of iodinium cation, which is assumed to decompose H2O2 oxidatively (6, 7). It has been believed that the halogenating activity of peroxidase has important roles in thyroid hormone synthesis (3, 11-13 ) and in biological defense mechanisms (11, 14, 15). Analyzing kinetically the iodinating reaction of thyroid peroxidase, we have concluded that the ratedetermining step is a transfer of the iodinium cation from the enzyme to tyrosine (3). When the iodinium cation acceptor is H2Oj, the products are O2 and iodide (6, 7). Several lines of evidence have indicated that enzymebound halogenium cations or liberated hypohalite ions play a bactericidal role (11, 14, 15). Woeber et al. (16) have reported that deiodination of thyroxine occurs during phagocytosis, being involved in the bactericidal action. Since we demonstrated that thyroid peroxidase selects 1 Present address: Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300, U.S.A. Abbreviations: T4, thyroxine; T,, 3,5,3'-triiodothyronine; T,, 3,5diiodothyronine; HPLC, high-performance liquid chromatography.
804
either one- or two-electron oxidations of iodotyrosines (10) and thyroglobulin (17) depending upon their iodine contents, we have been interested in the mechanism of iodothyronine oxidation catalyzed by thyroid peroxidase and lactoperoxidase. In this paper we report the relationship between catalatic activity induced by iodothyronines and oxidation of iodothyronines in the peroxidase systems. MATERIALS AND METHODS Thyroid peroxidase used was purified from hog thyroid microsomes by irnmunoaffinity chromatography (18). Lactoperoxidase was donated by Prof. Singo Nakamura, Hirosaki University, Hirosaki. The ratio of Am to AIM, was 0.24 for thyroid peroxidase and 0.92 for lactoperoxidase. The concentration of these peroxidases was calculated on the basis of a value of 114 for e mM at 413 nm (19). The O2 concentration was measured polarographically with a Clark-type 0 2 electrode. The iodide concentration was recorded with an Orion ionalyzer, model 811 equipped with an iodide electrode. The oxidation products of iodothyronines were analyzed with a Hitachi 655 HPLC system equipped with a Merck Lichrosorb RP-18 column. Stopped-flow measurements were performed using a Union Giken Rapid Reaction Analyzer, RA-1300. ESR spectra were recorded on a Varian E-109B spectrometer equipped with a stopped-flow apparatus. Diiodothyronine (T2), triiodothyronine (T,), and thyroxJ. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/108/5/804/813658 by Stockholm University Library user on 19 January 2019
Received for publication, May 25, 1990
805
Oxidation of Iodothyronines by Thyroid Peroxidase ine (T4) were obtained from Sigma (St. Louis). The other reagents used were obtained as described previously (3). The reactions were carried out in 0.1 M potassium phosphate (pH 7.4) at 20'C. RESULTS
B TPO
LPO
-EO+H 2 O
E + H2O2 -OH+H + T-O
n O T 3 >T 2 . To clarify the possibility that the iodothyronines serve as electron donors in the reactions of these peroxidases, kinetic experiments were carried out (Fig. 2). Figure 2, A and C, shows stopped-flow traces of peroxidase Compounds I and II during the reactions with iodothyronines. From the kinetic traces at various wavelengths, we obtained intermediate difference spectra of thyroid peroxidase and lactoperoxidase in the steady state of reactions (Fig. 2, B and D). Compound I was observed in the reactions of thyroid peroxidase with T2 and T3, and Compound II in the reaction with T4. Intermediate difference spectra of lactoperoxidase appearing during the oxidation of iodothyronines were invariably Compound II. The catalytic intermediates of the enzymes at the steady state and rate constants for ratedetermining steps of the reactions are summarized in Table I. The table suggested that thyroid peroxidase catalyzes the one-electron oxidation of T< and the two-electron oxidation of T3 and T2. It also shows that iodothyronines are better electron donors for these peroxidase reactions than iodotyrosines are (10). It has been reported that tyrosine and iodotyrosines are oxidized to dimerized products having characteristic UV spectra in the reactions of lactoperoxidase and thyroid peroxidase (21). Bj5rkste"n (22) reported that diiodotyrosine and iodide are detected after T4 is oxidized by a horseradish peroxidase system at pH 9. Therefore, the oxidation products of iodothyronines in the reactions of lactoperoxidase and thyroid peroxidase were analyzed by an HPLC method, which has been used for assay of T4 and
diiodotyrosine (23). However, no peak assigned to tyrosine or iodotyrosines appeared although T4, T3, and T2 completely disappeared 30 min after the reactions were initiated in the presence of about 2-fold molar excess of H2O2 to the iodothyronines (Fig. 3A). On the other hand, we could detect iodide liberation, which increased with the increase of H2O2 concentration at a fixed concentration of iodothyronines (Table II). The table also shows that the iodide liberation increased as the number of iodine atoms in iodothyronines increased. Even in the reaction with T 2 a significant amount of iodide was released. It has been reported that iodide is liberated not only from the 3'- and 5'-positions but also from the 3- and 5-positions of T4 by a horseradish peroxidase system (22). The iodide was also found to be released from diiodotyrosine in the same peroxidase systems (Table II). Since the catalatic activity of lactoperoxidase and thyroid peroxidase is enhanced by iodide ions, the iodothyroninesdependent catalatic activity of these peroxidases can be explained in terms of the iodide released from the iodothyronines in the peroxidase reaction. This explanation, however, was found to be too simplified as the iodothyronines could also accelerate the iodide-dependent catalatic reaction and in this case the order of the effect of iodothyronines was the reverse of that observed in Fig. 1 (Figs. 4 and 5). Under the experimental conditions in Figs. 4 and 5, the reaction of Compound I with iodide is so fast (24) that it may no longer be rate-limiting in the H2O2-decomposing reaction. The oxidation of iodothyronines was inhibited when the reactions were carried out in the presence of ascorbate or GSH (Fig. 3, B and C). We have already demonstrated phenol-mediated one- and two-electron oxidations of ascorbate and GSH in the peroxidase reactions (7). The iodothyronines also mediated the oxidation of ascorbate in the presence of the peroxidase systems as can be seen in Fig. 6. Although thyroid peroxidase and lactoperoxidase catalyzed the H2O2- dependent oxidation of ascorbate to some extent, the oxidation was greatly accelerated by the addition of iodothyronines. The oxidation rate was dependent upon the concentration of iodothyronines. From the initial velocity in T4 -, T3 -, and T2 - mediated ascorbate oxidation by thyroid peroxidase, the rate constants were estimated to be 2.3 x 105, 2.1 x 106, and 9.2 X105 M"1 • s"1. The rates were about the same as those estimated using the rate constants in Table I by assuming the following reactions. For thyroid peroxidase-T2 or T3-ascorbate (AH") systems (System I):
806
M. Nakamura ef
A
B 10s
. 430nm
"~420rvn
10s
4A0 460 Wavelength(nm)
460 Wavelength(nm)
and for thyroid peroxidase-T 4 -ascorbate and lactoperoxidase-T,, T3, or T 4 -ascorbate systems (System II): EO+T-OH EOH + T-OH - ^ T-O"+AH"
»EOH+T-O" E + T-0 + H,0
(4) (5)
> T-OH + A7
(6)
> A+AH-,
(7)
where T-OH, E, EO, and EOH denote iodothyronines, peroxidase, Compound I, and Compound II, respectively. In
the above two sequential reactions, rate constants for rate-limiting steps (r.l.s.) are shown in Table I. The difference in mechanism between the above two sequential reactions could be demonstrated in an ESR experiment. Figure 7 shows time courses of the ESR signal of ascorbate free radical during the peroxidase-catalyzed ascorbate oxidation in the presence or absence of iodothyronines. The addition of 25//M iodothyronines caused a considerable increase in the steady-state concentration of the radical as well as in the rate of ascorbate oxidation in the reactions of System II. The degree of the increase is compatible with the oxidation rate of iodothyronines by peroxidases (Table I). On the other hand, the addition of T3 J. Biochem.
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440 460 Wavelength(nm)
Fig. 2. Stopped-flow kinetics for the intermediates of thyroid peroxidase and lactoperozidase daring the reaction with iodothyronines. The reactions were carried out in the presence of 1.1 //M thyroid peroxidase or 0.9 //M lactoperoxidase, 5.5 fiM H,O2, and 25 fiM iodothyronine. A: Stopped-flow traces of thyroid peroxidase ai two wavelengths for T, (a), T, (b), and T, (c). The wavelength used indicates Compound II (430 nm) and Compound I (420 nm). B: Difference spectra of thyroid peroxidase during the oxidation of T, (a) and T4 (b). The spectra were obtained at the times indicated from stopped-flow traces at various wavelengths. C: Stopped-flow traces of lactoperoxidase at two wavelengths for T, (a), T, (b), and T4 (c). D: Difference spectra of lactoperoxidase during the oxidation of T, (a) and T, (b).
807
Oxidation of Iodothyronines by Thyroid Peroxidase A
TABLE II. Concentrations of iodide released from iodothyronines. Reaction mixtures contained 20 //M iodothyronines, various concentrations of H,O, and 25 nM thyroid peroxidase or 75 nM lactoperoxidase in the presence or absence of 10 //M KI. Iodide concentrations were measured after 20 min. H,O, KF I" released (//M) added G«M)
Thyroid peroxidase
Lactoperoxidase
20 80 80 20 80 80 20 80 80
0 0 10° 0 0 10* 0 0 101 0
5.0 8.3 12.0 3.3 4.1 8.6 1.2 2.0 6.7 3.9
5.1 7.1 11.0 3.4 4.5 8.1 0.7 2.1 7.0 3.1
T, 20 Retention time (min)
10
T,
B T,
Drr so (100 M ) Mir"
80
0
T 2 (Fig. 1), which is consistent with the amounts of iodide released (see Table II). In the presence of a suitable amount of iodide, the rate-limiting step is shifted from Reaction 8 to Reaction 9 and the effect of iodothyronines becomes greater in the order of T 2 >T 3 >T 4 (Fig. 5). We have reported that the transfer of iodinium cation from enzyme to acceptor is accelerated by iodothyronines, the effect being greater in the order of T 2 >T 3 >T 4 (3). The physiological meaning of peroxidase-catalyzed oxidation of iodothyronines is not clear. In this respect it might be worthwhile to point out the following three facts. 1) According to Klebanoff (14), iodide is more effective than chloride for the bactericidal activity of polymorphonuclear leucocytes, and it has been reported that iodothyronines play an important role in the myeloperoxidasemediated antimicrobial reaction (16). 2) No iodide release is observed when thyroglobulin containing a high level of iodine (0.7%) is oxidized by the thyroid peroxidase system (data not shown) even though the oxidation takes place through a free radical mechanism (17). 3) Thyroglobulin is known to be hormone-storing protein, for thyroid hormones are released from the molecule after selective cleavage in lysosomes (29-32). Although the thyroid hormones are readily oxidized by the thyroid peroxidase system, this oxidation is strongly inhibited by GSH and ascorbate, which are contained in the thyroid tissue in large quantities. REFERENCES 1. Thomas, J.A., Morris, D.R., & Hager, L.P. (1970) J. Biol Chem. 245, 3129-3134 2. Magnusson, R.P. & Taurog, A. (1983) Biochem. Biophys. Res. Commun. 112, 475-481 3. Nakamura, M., Yamazaki, I., Nakagawa, H., & Ohtaki, S. (1983) J. Biol Chem. 258, 3837-3842 4. Nakamura, M., Yamazaki, I., Nakagawa, H., Ohtaki, S., & Ui, N. (1984) J. Biol. Chem. 259, 359-364 5. Araiso, T., Rutter, R., Palcic, M.M., Hager, L.P., & Dunford, H. B. (1981) Can. J. Biochem. 59, 233-236 6. Magnusson, R.P., Taurog, A., & Dorris, MX. (1984) J. Biol. Chem. 259, 197-205 7. Nakamura, M., Yamazaki, I., Ohtaki, S., & Nakamura, S. (1986) J. Biol Chem. 261, 13923-13927 8. BjBrksten, F. (1970) Biochim Biophys. Acta 212, 396-406 9. Roman, R. & Dunford, H.B. (1972) Biochemistry 11, 2076-2082 10. Nakamura, M., Yamazaki, I., Kotani, T., & Ohtaki, S. (1985) J. Biol. Chem. 260, 13646-13552 11. Morrison, M. & Schonbaum, G. (1976) Annu. Rev. Biochem. 45, 861-888 12. Taurog, A. (1979) in Endocrinology (DeGroot, L.J., Cahill, G.F., Jr., Odell, W.D., Martini, L., Potts, J.T., Jr., Nelson, D.G., Steinberger, E., & Winegrad, A.I., eds.) Vol. 1. pp. 331-342, Grune and Stratton, New York 13. Nunez, J. (1988) in The Thyroid Gland (De Visscher, M., ed.) pp. 39-59, Raven Press, New York 14. Klebanoff, S.J. (1967) J. Exp. Med. 126, 1063-1079 15. Thomas, E.L. & Aune, T.M. (1978) Antimicrob. Agents Chemother.13, 1000-1005 16. Woeber, K.A., Doherty, G.F., &Ingbar, S.H. (1972) Science 176, 1039-1041
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TABLE HI. Decomposition of iodothyronines by thyroid peroxidase (A) and lactoperoxidase (B) in the presence or absence of iodide. Reaction mixtures contained 25 nM thyroid peroxidase or 75 nM lactoperoxidase, 20 fiM iodothyronines and various concentrations of H,Oi in the presence or absence of 20/iM KI. Aliquots were withdrawn for assay after 30 min. Concentrations of iodothyronines were determined by the use of HPLC according to Alexander and Nishimoto (23). KI H,O, T. T, T, Iodothyronines added added 0/M) 0/M) 1.4 0 0 0 40 A, T, 0 20 0 40 18.5
809
810
26. Borg, D.C. (1972) in Biological Applications of Electron Spin Resonance (Swartx, H.M., Bolton, J.B., & Borg, D.C., eds.) pp. 265-350, John Wiley and Sons, New York 27. Rosenberg, L.L., Nataf, B.M., & Cavalieri, R.R. (1973) Endocrinology 93, 1066-1076 28. VanZyl, A., Dorris, M.L., &Taurog, A. (1974) Endocrinology95, 1166-1173 29. DeGroot, L.J., & Taurog, A. (1979) in Endocrinology (DeGroot, L.J., Cahill, G.F., Jr., Odell, W.D., Martini, L., Potte, J.T., Jr., Nelson, D.G., Steinberger, E., & Winegrad, A.I., eds.) Vol. 1, pp. 343-346. Grune and Stratton, New York 30. Nakagawa, H. & Ohtaki, S. (1984) Endocrinology 115, 33-40 31. Nakagawa, H. & Ohtaki, S. (1985) Endocrinology 116, 14331439 32. Rousset, B., Selmi, S., Bornet, H., Bourgeat, P., Rabilloud, R, & Munari-Silem, Y. (1989) J. BioL Chem. 264, 12620-12626
J. Biochem.
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17. Nakamura, M., Yamazaki, I., Kotani, T., & Ohtaki, S. (1989) J. BioL Chan. 264, 12909-12913 18. Nakagawa, H., Kotani, T., Ohtaki, S., Nakamura, M., & Yamalaki, I. (1985) Biochem. Biophys. Res. Commun. 127, 8-14 19. Ohtaki, S., Nakagawa, H., Nakamura, S., Nakamura, M., & Yamaiaki, I. (1985) J. BioL Chan. 260, 441-448 20. Chance, B. (1957) Arch. Biochem. Biophys. 71, 130-136 21. Ohtaki, S., Nakagawa, H., Nakamura, M., & Yamazaki, I. (1982) J. BioL Chan. 267, 13398-13403 22. BjBrksten, F. (1966) Ada Chem. Scand. 5, 1438-1439 23. Alexander, N.M. & Nishimoto, M. (1979) Clin. Chem. 25, 17571760 24. Ohtaki, S., Nakagawa, H., Kimura, S., & Yamaiaki, I. (1981) J. BioL Chem. 256, 805-810 25. Ross, D., Norbeck, K., & Moldeus, P. (1985) J. BioL Chem. 260, 15028-15032
M. Nakamura et aL
Iodothyronine-Induced Catalatic Activity of Thyroid Peroxidase Masao Nakamura,* Isao Yamazaki,*1' and Sachiya Ohtaki** 'Biophysics Division, Research Institute of Applied Electricity, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060; and *' Central Laboratory of Clinical Investigation, Miyazaki Medical College Hospital, Kiyotake, Miyazaki 889-16
Iodothyronines induced catalatic (H202-decomposing) activity of thyroid peroxidase and lactoperoxidase, the effect increasing in the order of thyroxine (T4)>triiodothyronine (T3) > diiodothyronine (T2). The iodothyronines served as electron donors in the peroxidase reactions, and during the reactions the catalytic intermediate of thyroid peroxidase was confirmed to be Compound II for T4 and Compound I for T3 and T2 and from the Soret absorption spectra obtained by stopped-flow measurements. Rate constants for the reactions between T4 and Compound II, T3 and Compound I, and T2 and Compound I were estimated at 1.9X 10s,1.3X 106, and 7.1 X10s M 1 - s 1 , respectively. Unlike the case of thyroid peroxidase, the catalytic intermediate of lactoperoxidase observed during the oxidation of iodothyronines was invariably Compound II. From these and other data it was concluded that thyroid peroxidase catalyzed one-electron oxidation of T4 and two-electron oxidations of T2 and T3 while lactoperoxidase catalyzed exclusively one-electron oxidation of the iodothyronines. Iodide was released during the enzymatic oxidation of iodothyronines, irrespective of the mechanism of one-electron and two-electron oxidations. The amount of released iodide increased in the order of T4 >T3 >T2. The iodothyronines-induced catalatic activity of these peroxidases was ascribable to the release of iodide, but it was also found that the iodide-enhanced catalatic activity was stimulated by iodothyronines. In this case the effect of iodothyronines was greater in the order of T2 >T3 >T4, which was consistent with the order of iodothyronine activation for the iodinium cation transfer from enzyme to acceptor.
It has been demonstrated that peroxidases have more or less catalatic (H2O2-decomposing) activity (1-6), which increases in the presence of halides (1, 2, 6, 7). In particular, thyroid peroxidase and lactoperoxidase exhibit a strong iodide-dependent catalatic activity (2, 7). The two-electron oxidation of iodide has been confirmed in the reactions of horseradish peroxidase (8, 9), lactoperoxidase, and thyroid peroxidase (10), resulting in the formation of iodinium cation, which is assumed to decompose H2O2 oxidatively (6, 7). It has been believed that the halogenating activity of peroxidase has important roles in thyroid hormone synthesis (3, 11-13 ) and in biological defense mechanisms (11, 14, 15). Analyzing kinetically the iodinating reaction of thyroid peroxidase, we have concluded that the ratedetermining step is a transfer of the iodinium cation from the enzyme to tyrosine (3). When the iodinium cation acceptor is H2Oj, the products are O2 and iodide (6, 7). Several lines of evidence have indicated that enzymebound halogenium cations or liberated hypohalite ions play a bactericidal role (11, 14, 15). Woeber et al. (16) have reported that deiodination of thyroxine occurs during phagocytosis, being involved in the bactericidal action. Since we demonstrated that thyroid peroxidase selects 1 Present address: Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300, U.S.A. Abbreviations: T4, thyroxine; T,, 3,5,3'-triiodothyronine; T,, 3,5diiodothyronine; HPLC, high-performance liquid chromatography.
804
either one- or two-electron oxidations of iodotyrosines (10) and thyroglobulin (17) depending upon their iodine contents, we have been interested in the mechanism of iodothyronine oxidation catalyzed by thyroid peroxidase and lactoperoxidase. In this paper we report the relationship between catalatic activity induced by iodothyronines and oxidation of iodothyronines in the peroxidase systems. MATERIALS AND METHODS Thyroid peroxidase used was purified from hog thyroid microsomes by irnmunoaffinity chromatography (18). Lactoperoxidase was donated by Prof. Singo Nakamura, Hirosaki University, Hirosaki. The ratio of Am to AIM, was 0.24 for thyroid peroxidase and 0.92 for lactoperoxidase. The concentration of these peroxidases was calculated on the basis of a value of 114 for e mM at 413 nm (19). The O2 concentration was measured polarographically with a Clark-type 0 2 electrode. The iodide concentration was recorded with an Orion ionalyzer, model 811 equipped with an iodide electrode. The oxidation products of iodothyronines were analyzed with a Hitachi 655 HPLC system equipped with a Merck Lichrosorb RP-18 column. Stopped-flow measurements were performed using a Union Giken Rapid Reaction Analyzer, RA-1300. ESR spectra were recorded on a Varian E-109B spectrometer equipped with a stopped-flow apparatus. Diiodothyronine (T2), triiodothyronine (T,), and thyroxJ. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/108/5/804/813658 by Stockholm University Library user on 19 January 2019
Received for publication, May 25, 1990
805
Oxidation of Iodothyronines by Thyroid Peroxidase ine (T4) were obtained from Sigma (St. Louis). The other reagents used were obtained as described previously (3). The reactions were carried out in 0.1 M potassium phosphate (pH 7.4) at 20'C. RESULTS
B TPO
LPO
-EO+H 2 O
E + H2O2 -OH+H + T-O
n O T 3 >T 2 . To clarify the possibility that the iodothyronines serve as electron donors in the reactions of these peroxidases, kinetic experiments were carried out (Fig. 2). Figure 2, A and C, shows stopped-flow traces of peroxidase Compounds I and II during the reactions with iodothyronines. From the kinetic traces at various wavelengths, we obtained intermediate difference spectra of thyroid peroxidase and lactoperoxidase in the steady state of reactions (Fig. 2, B and D). Compound I was observed in the reactions of thyroid peroxidase with T2 and T3, and Compound II in the reaction with T4. Intermediate difference spectra of lactoperoxidase appearing during the oxidation of iodothyronines were invariably Compound II. The catalytic intermediates of the enzymes at the steady state and rate constants for ratedetermining steps of the reactions are summarized in Table I. The table suggested that thyroid peroxidase catalyzes the one-electron oxidation of T< and the two-electron oxidation of T3 and T2. It also shows that iodothyronines are better electron donors for these peroxidase reactions than iodotyrosines are (10). It has been reported that tyrosine and iodotyrosines are oxidized to dimerized products having characteristic UV spectra in the reactions of lactoperoxidase and thyroid peroxidase (21). Bj5rkste"n (22) reported that diiodotyrosine and iodide are detected after T4 is oxidized by a horseradish peroxidase system at pH 9. Therefore, the oxidation products of iodothyronines in the reactions of lactoperoxidase and thyroid peroxidase were analyzed by an HPLC method, which has been used for assay of T4 and
diiodotyrosine (23). However, no peak assigned to tyrosine or iodotyrosines appeared although T4, T3, and T2 completely disappeared 30 min after the reactions were initiated in the presence of about 2-fold molar excess of H2O2 to the iodothyronines (Fig. 3A). On the other hand, we could detect iodide liberation, which increased with the increase of H2O2 concentration at a fixed concentration of iodothyronines (Table II). The table also shows that the iodide liberation increased as the number of iodine atoms in iodothyronines increased. Even in the reaction with T 2 a significant amount of iodide was released. It has been reported that iodide is liberated not only from the 3'- and 5'-positions but also from the 3- and 5-positions of T4 by a horseradish peroxidase system (22). The iodide was also found to be released from diiodotyrosine in the same peroxidase systems (Table II). Since the catalatic activity of lactoperoxidase and thyroid peroxidase is enhanced by iodide ions, the iodothyroninesdependent catalatic activity of these peroxidases can be explained in terms of the iodide released from the iodothyronines in the peroxidase reaction. This explanation, however, was found to be too simplified as the iodothyronines could also accelerate the iodide-dependent catalatic reaction and in this case the order of the effect of iodothyronines was the reverse of that observed in Fig. 1 (Figs. 4 and 5). Under the experimental conditions in Figs. 4 and 5, the reaction of Compound I with iodide is so fast (24) that it may no longer be rate-limiting in the H2O2-decomposing reaction. The oxidation of iodothyronines was inhibited when the reactions were carried out in the presence of ascorbate or GSH (Fig. 3, B and C). We have already demonstrated phenol-mediated one- and two-electron oxidations of ascorbate and GSH in the peroxidase reactions (7). The iodothyronines also mediated the oxidation of ascorbate in the presence of the peroxidase systems as can be seen in Fig. 6. Although thyroid peroxidase and lactoperoxidase catalyzed the H2O2- dependent oxidation of ascorbate to some extent, the oxidation was greatly accelerated by the addition of iodothyronines. The oxidation rate was dependent upon the concentration of iodothyronines. From the initial velocity in T4 -, T3 -, and T2 - mediated ascorbate oxidation by thyroid peroxidase, the rate constants were estimated to be 2.3 x 105, 2.1 x 106, and 9.2 X105 M"1 • s"1. The rates were about the same as those estimated using the rate constants in Table I by assuming the following reactions. For thyroid peroxidase-T2 or T3-ascorbate (AH") systems (System I):
806
M. Nakamura ef
A
B 10s
. 430nm
"~420rvn
10s
4A0 460 Wavelength(nm)
460 Wavelength(nm)
and for thyroid peroxidase-T 4 -ascorbate and lactoperoxidase-T,, T3, or T 4 -ascorbate systems (System II): EO+T-OH EOH + T-OH - ^ T-O"+AH"
»EOH+T-O" E + T-0 + H,0
(4) (5)
> T-OH + A7
(6)
> A+AH-,
(7)
where T-OH, E, EO, and EOH denote iodothyronines, peroxidase, Compound I, and Compound II, respectively. In
the above two sequential reactions, rate constants for rate-limiting steps (r.l.s.) are shown in Table I. The difference in mechanism between the above two sequential reactions could be demonstrated in an ESR experiment. Figure 7 shows time courses of the ESR signal of ascorbate free radical during the peroxidase-catalyzed ascorbate oxidation in the presence or absence of iodothyronines. The addition of 25//M iodothyronines caused a considerable increase in the steady-state concentration of the radical as well as in the rate of ascorbate oxidation in the reactions of System II. The degree of the increase is compatible with the oxidation rate of iodothyronines by peroxidases (Table I). On the other hand, the addition of T3 J. Biochem.
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440 460 Wavelength(nm)
Fig. 2. Stopped-flow kinetics for the intermediates of thyroid peroxidase and lactoperozidase daring the reaction with iodothyronines. The reactions were carried out in the presence of 1.1 //M thyroid peroxidase or 0.9 //M lactoperoxidase, 5.5 fiM H,O2, and 25 fiM iodothyronine. A: Stopped-flow traces of thyroid peroxidase ai two wavelengths for T, (a), T, (b), and T, (c). The wavelength used indicates Compound II (430 nm) and Compound I (420 nm). B: Difference spectra of thyroid peroxidase during the oxidation of T, (a) and T4 (b). The spectra were obtained at the times indicated from stopped-flow traces at various wavelengths. C: Stopped-flow traces of lactoperoxidase at two wavelengths for T, (a), T, (b), and T4 (c). D: Difference spectra of lactoperoxidase during the oxidation of T, (a) and T, (b).
807
Oxidation of Iodothyronines by Thyroid Peroxidase A
TABLE II. Concentrations of iodide released from iodothyronines. Reaction mixtures contained 20 //M iodothyronines, various concentrations of H,O, and 25 nM thyroid peroxidase or 75 nM lactoperoxidase in the presence or absence of 10 //M KI. Iodide concentrations were measured after 20 min. H,O, KF I" released (//M) added G«M)
Thyroid peroxidase
Lactoperoxidase
20 80 80 20 80 80 20 80 80
0 0 10° 0 0 10* 0 0 101 0
5.0 8.3 12.0 3.3 4.1 8.6 1.2 2.0 6.7 3.9
5.1 7.1 11.0 3.4 4.5 8.1 0.7 2.1 7.0 3.1
T, 20 Retention time (min)
10
T,
B T,
Drr so (100 M ) Mir"
80
0
T 2 (Fig. 1), which is consistent with the amounts of iodide released (see Table II). In the presence of a suitable amount of iodide, the rate-limiting step is shifted from Reaction 8 to Reaction 9 and the effect of iodothyronines becomes greater in the order of T 2 >T 3 >T 4 (Fig. 5). We have reported that the transfer of iodinium cation from enzyme to acceptor is accelerated by iodothyronines, the effect being greater in the order of T 2 >T 3 >T 4 (3). The physiological meaning of peroxidase-catalyzed oxidation of iodothyronines is not clear. In this respect it might be worthwhile to point out the following three facts. 1) According to Klebanoff (14), iodide is more effective than chloride for the bactericidal activity of polymorphonuclear leucocytes, and it has been reported that iodothyronines play an important role in the myeloperoxidasemediated antimicrobial reaction (16). 2) No iodide release is observed when thyroglobulin containing a high level of iodine (0.7%) is oxidized by the thyroid peroxidase system (data not shown) even though the oxidation takes place through a free radical mechanism (17). 3) Thyroglobulin is known to be hormone-storing protein, for thyroid hormones are released from the molecule after selective cleavage in lysosomes (29-32). Although the thyroid hormones are readily oxidized by the thyroid peroxidase system, this oxidation is strongly inhibited by GSH and ascorbate, which are contained in the thyroid tissue in large quantities. REFERENCES 1. Thomas, J.A., Morris, D.R., & Hager, L.P. (1970) J. Biol Chem. 245, 3129-3134 2. Magnusson, R.P. & Taurog, A. (1983) Biochem. Biophys. Res. Commun. 112, 475-481 3. Nakamura, M., Yamazaki, I., Nakagawa, H., & Ohtaki, S. (1983) J. Biol Chem. 258, 3837-3842 4. Nakamura, M., Yamazaki, I., Nakagawa, H., Ohtaki, S., & Ui, N. (1984) J. Biol. Chem. 259, 359-364 5. Araiso, T., Rutter, R., Palcic, M.M., Hager, L.P., & Dunford, H. B. (1981) Can. J. Biochem. 59, 233-236 6. Magnusson, R.P., Taurog, A., & Dorris, MX. (1984) J. Biol. Chem. 259, 197-205 7. Nakamura, M., Yamazaki, I., Ohtaki, S., & Nakamura, S. (1986) J. Biol Chem. 261, 13923-13927 8. BjBrksten, F. (1970) Biochim Biophys. Acta 212, 396-406 9. Roman, R. & Dunford, H.B. (1972) Biochemistry 11, 2076-2082 10. Nakamura, M., Yamazaki, I., Kotani, T., & Ohtaki, S. (1985) J. Biol. Chem. 260, 13646-13552 11. Morrison, M. & Schonbaum, G. (1976) Annu. Rev. Biochem. 45, 861-888 12. Taurog, A. (1979) in Endocrinology (DeGroot, L.J., Cahill, G.F., Jr., Odell, W.D., Martini, L., Potts, J.T., Jr., Nelson, D.G., Steinberger, E., & Winegrad, A.I., eds.) Vol. 1. pp. 331-342, Grune and Stratton, New York 13. Nunez, J. (1988) in The Thyroid Gland (De Visscher, M., ed.) pp. 39-59, Raven Press, New York 14. Klebanoff, S.J. (1967) J. Exp. Med. 126, 1063-1079 15. Thomas, E.L. & Aune, T.M. (1978) Antimicrob. Agents Chemother.13, 1000-1005 16. Woeber, K.A., Doherty, G.F., &Ingbar, S.H. (1972) Science 176, 1039-1041
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TABLE HI. Decomposition of iodothyronines by thyroid peroxidase (A) and lactoperoxidase (B) in the presence or absence of iodide. Reaction mixtures contained 25 nM thyroid peroxidase or 75 nM lactoperoxidase, 20 fiM iodothyronines and various concentrations of H,Oi in the presence or absence of 20/iM KI. Aliquots were withdrawn for assay after 30 min. Concentrations of iodothyronines were determined by the use of HPLC according to Alexander and Nishimoto (23). KI H,O, T. T, T, Iodothyronines added added 0/M) 0/M) 1.4 0 0 0 40 A, T, 0 20 0 40 18.5
809
810
26. Borg, D.C. (1972) in Biological Applications of Electron Spin Resonance (Swartx, H.M., Bolton, J.B., & Borg, D.C., eds.) pp. 265-350, John Wiley and Sons, New York 27. Rosenberg, L.L., Nataf, B.M., & Cavalieri, R.R. (1973) Endocrinology 93, 1066-1076 28. VanZyl, A., Dorris, M.L., &Taurog, A. (1974) Endocrinology95, 1166-1173 29. DeGroot, L.J., & Taurog, A. (1979) in Endocrinology (DeGroot, L.J., Cahill, G.F., Jr., Odell, W.D., Martini, L., Potte, J.T., Jr., Nelson, D.G., Steinberger, E., & Winegrad, A.I., eds.) Vol. 1, pp. 343-346. Grune and Stratton, New York 30. Nakagawa, H. & Ohtaki, S. (1984) Endocrinology 115, 33-40 31. Nakagawa, H. & Ohtaki, S. (1985) Endocrinology 116, 14331439 32. Rousset, B., Selmi, S., Bornet, H., Bourgeat, P., Rabilloud, R, & Munari-Silem, Y. (1989) J. BioL Chem. 264, 12620-12626
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/108/5/804/813658 by Stockholm University Library user on 19 January 2019
17. Nakamura, M., Yamazaki, I., Kotani, T., & Ohtaki, S. (1989) J. BioL Chan. 264, 12909-12913 18. Nakagawa, H., Kotani, T., Ohtaki, S., Nakamura, M., & Yamalaki, I. (1985) Biochem. Biophys. Res. Commun. 127, 8-14 19. Ohtaki, S., Nakagawa, H., Nakamura, S., Nakamura, M., & Yamaiaki, I. (1985) J. BioL Chan. 260, 441-448 20. Chance, B. (1957) Arch. Biochem. Biophys. 71, 130-136 21. Ohtaki, S., Nakagawa, H., Nakamura, M., & Yamazaki, I. (1982) J. BioL Chan. 267, 13398-13403 22. BjBrksten, F. (1966) Ada Chem. Scand. 5, 1438-1439 23. Alexander, N.M. & Nishimoto, M. (1979) Clin. Chem. 25, 17571760 24. Ohtaki, S., Nakagawa, H., Kimura, S., & Yamaiaki, I. (1981) J. BioL Chem. 256, 805-810 25. Ross, D., Norbeck, K., & Moldeus, P. (1985) J. BioL Chem. 260, 15028-15032
M. Nakamura et aL
