ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 296, No. 1, July, pp. 239-246, 1992
Myeloperoxidase-Catalyzed Alvin Taurog2 and Martha Department
of Pharmacology,
Received November
lodination
L. Dorris
University
of Texas Southwestern
Press, Inc.
‘ This work was supported by research grants from Boots Pharmaceuticals and from the United States Public Health Service (NIDDK03612). ’ TO whom correspondence should be addressed.
Thyroid peroxidase (TP0)3, an enzyme that plays an essential role in the formation of thyroid hormones, catalyzes both iodination of thyroglobulin and the intramolecular coupling of two diiodotyrosine (DIT) molecules to form thyroxine (T4) and of one monoiodotyrosine (MIT) molecule and one DIT molecule to form 3’,3,5triiodothyronine (T3) (1). We previously observed (2) that lactoperoxidase (LPO), an enzyme isolated from milk, also catalyzes these reactions with much the same efficiency as TPO. Both purified TPO and LPO have been employed in model systems to study mechanisms of peroxidase-catalyzed iodination and coupling (1). More recently, the complete primary amino acid sequences of porcine (3) and human (4-6) TPO, human myeloperoxidase (MPO) (7,8), and bovine LPO (9) have been deduced from cDNA cloning experiments. These sequences display considerable homology, especially in regions considered to be important for binding the heme prosthetic group (10). Although myeloperoxidase (MPO) is known to catalyze iodination reactions (11-S), very little has been reported regarding its ability to catalyze iodination of thyroglobulin (Tg). The sequence similarities with TPO and LPO, together with our previous studies demonstrating that LPO as well as TPO catalyzes the coupling reaction, suggestedthat MPO also might catalyze T4 biosynthesis in Tg. It was the purpose of this study to investigate this possibility, and also to examine the effect of pH and of chloride on MPO-catalyzed iodination of Tg. METHODS
AND
MATERIALS
Human MPO was purchased from Alpha TheraMyeloperoxidase. peutic Corp. (Los Angeles, CA). The lyophilized product, containing a high concentration of salt, was dialyzed against 67 mM phosphate buffer, pH 7.0, and aliquots of the frozen solution were stored at -20°C. The value for Aao/Am was 0.74, and enzyme concentration was calculated
3 Abbreviations used: MPO, myeloperoxiclase; TPO, thyroid peroxidase; LPO, lactoperoxidase; HRP, horseradish peroxidase; Tg, thyroglobulin; T*, thyroxine; Ts, 3’,3,5-triiodothyronine; DIT, diiodotyrosine; MIT, monoiodotyrosine; MMI, 1-methyl-2-mercaptoimidazole; PTU, 6-propyl-2-thiouracih BSA, bovine serum albumin.
239
$5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction
Medical Center, Dallas, Texas 75235
8,1991, and in revised form March lo,1992
Myeloperoxidase (MPO), which displays considerable amino acid sequence homology with thyroid peroxidase (TPO) and lactoperoxidase (LPO), was tested for its ability to catalyze iodination of thyroglobulin and coupling of two diiodotyrosyl residues within thyroglobulin to form thyroxine. After 1 min of incubation in a system containing goiter thyroglobulin, II, and HzOz, the pH optimum of MPO-catalyzed iodination was markedly acidic (-4.0), compared to LPO (-5.4) and TPO (-6.6). The presence of 0.1 N Cl- or Br- shifted the pH optimum for MPO to about 5.4 but had little or no effect on TPO- or LPO-catalyzed iodination. At pH 5.4, 0.1 N Cl- and 0.1 N Br- had a marked stimulatory effect on MPO-catalyzed iodination. At pH 4.0, however, iodinating activity of MPO was almost completely inhibited by 0.1 N Cl- or Br-. Inhibition of chlorinating activity of MPO by Cl- at pH 4.0 has been previously described. When iodination of goiter thyroglobulin was performed with MPO plus the HzOz generating system, glucose-glucose oxidase, at pH 7.0, the iodinating activity was markedly increased iodination and thyby 0.1 N Cl-. Under these conditions roxine formation were comparable to values observed with TPO. MPO and TPO were also compared for coupling activity in a system that measures coupling of diiodotyrosyl residues in thyroglobulin in the absence of iodination. MPO displayed very significant coupling activity, and, like TPO, this activity was stimulated by a low concentration of free diiodotyrosine (1 FM). The thioureylene drugs, propylthiouracil and methimazole, inhibited MPO-catalyzed iodination both reversibly and irreversibly, in a manner similar to that previously described for TPO-catalyzed iodination. o 1992 Academic
0003-9861/92
and Coupling’
in any form reserved.
240
TAUROG
AND
on the basis of a millimolar extinction coefficient at 430 nm of 178 (16, 17). Some experiments were performed with bovine MPO, purchased from ExOxEmis (San Antonio, TX), after human MPO became unavailable from our original supplier. The bovine MPO displayed a value of 0.72 for A,,O/Azso, and based on our guaiacol assay it was about 17% more active (per mole of heme) than the human MPO. Lactoperoxidase. Bovine LPO was obtained from Pharmacia or Sigma. It was dialyzed against 67 mu phosphate, pH 7.0, and the enzyme concentration was based on a millimolar extinction coefficient at 412 nm of 114 (18). The value for A412/A280was 0.89-0.90. Thyroid peroxidase. Porcine TPO (Preparation XIII) was prepared in this laboratory as previously described (19, 20). It is a highly active, trypsin-detergent-solubilized product, whose relationship to the native enzyme has been characterized (20). The value for Ad12/Am was 0.48. The extinction coefficient for TPO has not been accurately determined, and in this study it was assumed to be the same as that for LPO. Goiter thyroglobulin. Thyroglobulin (Tg) was isolated from a large goitrous human thyroid and purified by repeated gel filtration through Bio-Gel A-5m (21). The final lyophilized material contained 0.038% I, equivalent to an average of two atoms of I per molecule. The concentration of Tg was determined by measurement of A*, using an extinction coefficient of 10.0 for a 1% solution (22). Reagents. BSA (crystallized) was obtained from ICN Biochemicals, glucose oxidase (lyophilized, 100 U/mg) from Boehringer Mannheim, PTU and MM1 from Aldrich Chemical Co., and NaOCl (analytical reagent) from Mallinckrodt Chemical CO. Zodinution procedure. The incubation mixture contained goiter Tg or BSA, peroxidase, radiolabeled iodide, and either directly added HzOz or the H202 generating system, glucose-glucose oxidase. The concentrations of the various components are provided with the results of individual experiments. In experiments designed to study the effect of pH, the pH was varied from 3.5 to 7.5. Phosphate buffer (67 mM) was used for pH 5.4 and above, and acetate buffer (0.1 M) for pHs below 5.4. The incubation mixture in many cases contained 0.1 N NaCl, and in some cases 0.1 N NaBr. We observed that the presence of 0.1 N Cl- or 0.1 N Br- in phosphate buffers decreased the measured pH by 0.1-0.2 pH units. A decrease was also observed with acetate buffer, but of smaller magnitude. These results are consistent with the known effect of ionic strength on the pK,, of neutral and anionic acids (23). Measured pH values are shown in the figures presented in the Results section. Experiments with added HzOz were used to approximate initial rates of iodination. The reaction was initiated with H,Oz and stopped after 1 min by addition of a large excess of I-methyl-2-mercaptoimidazole (MMI, methimazole, final concentration, 5 mM). Protein-bound iodine was separated from unreacted iodide by paper chromatography in collidine-NH,OH, as previously described (24). Results are expressed as pM I- bound to protein in 1 min. For measurements of T4 formation in Tg iodinated with MPO or TPO, the iodination was performed with H,Os generated by glucoseglucose oxidase. Under these conditions the degree of iodination increased steadily for at least 30 min. The reaction was initiated with glucose oxidase and stopped with methimazole after a 45-min incubation at 37°C. The Tg was digested with pronase plus aminopeptidase, and labeled T1, Ts. MIT, and DIT were separated by paper chromatography, as previously described (25). The corresponding sections of the chromatogram, visualized by radioautography, were counted, and residues of T4, T3, MIT, and DIT were calculated from the degree of iodination and the radioiodine distribution. Coupling with chemically labeled Tg. Goiter Tg was iodinated chemically with radiolabeled 1, to a level of 23-29 atoms I per molecule. The radiolabeled 1; solution contained 7.07 mM IZ (determined by titration with standard thiosulfate) in 26 mM Kl and a very small volume of iz61
DORRIS
or i3iI, added to yield a specific activity of about 100 or 200 pCi/geq I, respectively. The labeled I; was added to 5-10 ml of a magnetically stirred solution of 1.5 ELMTg (7 ~1 of I; per ml of Tg), and after 2 min of stirring, 10 ~1 of 1 M sodium thiosulfate was added to reduce unreacted I;. The number of iodine atoms bound per molecule of Tg was calculated from the amount of radioiodine remaining at the origin of the paper chromatogram (protein-bound), the specific activity of the iodine in the labeled 1, solution, and the protein concentration. The labeled Tg solution was dialyzed thoroughly against 67 mM phosphate pH 7.0 at 4°C (400 vol, two changes of buffer) over a period of 22 h to remove inorganic radioiodine. After dialysis, the labeled Tg, which contained almost all of its radioiodine as DIT and MIT, and very little as Tq, was incubated with MPO or TPO under the following conditions: 1.5 pM Tg, 25 nM MPO or TPO, 1 mg/ml glucose, and 0.1 pg/ml glucose oxidase, in 65 mM phosphate, pH 7.0, for 20 min at 37°C. The reaction was stopped with excess methimazole, and the incubation mixture was digested with pronase plus aminopeptidase and the labeled iodoamino acid distribution determined as previously described (25). Coupling activity was measured by the increase in the percentage of radioiodine present as T4 + Ts, compared to the percentages in labeled Tg incubated in the same system lacking peroxidase. Zodination of BSA andgoiter Tg with NaOClplus Z-. Results obtained in the present study with MPO-catalyzed iodination at pH 5.4 in the presence of 0.1 N Cl- suggested that HOC1 might be an intermediate in the iodination reaction. To test this possibility we used a solution of NaOCl for iodination of BSA and goiter Tg. The NaOCl was standardized by iodimetric titration. The incubation mixture contained either 7.5 PM BSA or 1 pM goiter Tg, 100 PM izr’I-, and 300 PM NaOCl, in phosphate buffer, pH 5.4. The reaction was initiated with the NaOCl and was terminated after 1, 3, or 5 min by the addition of sodium thiosulfate (final concentration, 10 mM). Protein-bound ‘%I was determined by paper chromatography, as described above. Inhibition of MPO-catalyzed iodination by 6-propylthiouracil (PTU) and I-methyl-2-mercaptoimidazole (MMZ). The incubation system contained 25 nM bovine MPO, 7.5 PM BSA, 100 pM i311-, 0.1 N NaCl, 1 mg/ml glucose, and 0.5 pg/ml glucose oxidase, in 60 mM phosphate buffer, pH 7.0. Drug concentrations varied from 0 to 300 FM. After initiation of the reaction at 37’C with glucose oxidase, samples were removed at 3,10,25,45, and 65 min and added to small tubes containing a small volume of 0.5 M MM1 to stop the reaction. The tubes were placed in an ice bath at O”C, and 25 ~1 of the reaction mixture was applied for filter paper chromatography in co&dine-NH,OH to determine the fraction of the 1311bound to protein.
RESULTS Effect of pH and Cl- on MPO-, TPO-, and LPO-catalyzed iodination of goiter Tg. Figure 1 shows results obtained after 1 min of incubation in a system containing MPO, TPO, or LPO, goiter Tg, labeled iodide, and H202 at varying pH and in the presence and absence of 0.1 N Cl-. Results are expressed as PM I- bound in 1 min. MPO readily catalyzed iodination of Tg in a pH- and Cl-dependent manner. In the absence of Cl-, the pH optimum of the reaction was about 4.0, and under the conditions of the incubation 2.3 X lo3 mol of I- was bound in 1 min per mole of enzyme. In the presence of 0.1 N Cl-, the pH optimum shifted to about 5.4, and 2.1 X lo3 mol of I- was bound in 1 min per mole of enzyme. At pH 4.0, 0.1 N Cl- was a potent inhibitor of iodination. This may be attributed to an inhibitory binding site for Cl- on
MYELOPEROXIDASE-CATALYZED
2 28 .E E t .E 24 z 3
20-
i
16-
100 ;M H202 or 0.1 N Cl-
I0
IODINATION
AND
241
COUPLING
in Fig. 1, iodinating activity catalyzed by MPO was significantly stimulated in the presence of 0.1 N Cl-. After a 30-min incubation, 79% of the I- was bound to Tg, compared to 50% in the absence of Cl-. When 0.1 N Cl- was present in the incubation mixture, MPO catalyzed the iodination of Tg as readily as did TPO (Fig. 2). However, in contrast to its effect on MPO-catalyzed iodination, Clhad no effect on TPO-catalyzed iodination (Fig. 2), or on LPO-catalyzed iodination (results not shown).
I
Iodoamino acid distribution in Tg iodinated with the MPO-glucose-glucose oxidase system. Results of two
12a-
1
5.0
I
5.5 PH
I
I
1
I
6.0
6.5
7.0
7.5
FIG. 1. Peroxidase-catalyzed iodination of goiter Tg at various pHs with and without 0.1 N Cl-; comparison of MPO, TPO, and LPO. The reaction was initiated at 37°C by addition of Hz02 and stopped after 1 min by addition of methimazole (final concn. 5 mM). Phosphate buffer (67 mM) was used for pH 5.4 and above and acetate buffer (0.1 N) for decreased pH pHs below pH 5.4. Addition of 0.1 N Cl- significantly values, especially in phosphate buffers.
MPO, which, as previously described (26), requires prior protonation. Iodination of goiter Tg catalyzed by TPO and by LPO under exactly the same conditions used for MPO was also highly pH-dependent but showed much less effect of Cl-. The pH optimum for TPO was about 6.6, and this was unaffected by 0.1 N Cl-. The peak activity of TPO was 2.7 X lo3 mol of I- bound per mole of enzyme, and this was slightly reduced in the presence of 0.1 N Cl-. LPO displayed maximum activity at about pH 5.4, and under the conditions used it was the most active of the peroxidases, binding 4.3 X lo3 mol of I- per mole of enzyme in one min. The pH optimum and the activity of LPO were little affected by 0.1 N Cl-. MPO-catalyzed iodination of goiter Tg at pH 7.0 with Hz02 generated by glucose-glucose oxidase; effect of 0.1 N
Cl-. The time course of MPO-catalyzed iodination of Tg in an incubation system containing the HzOz generating system, glucose-glucose oxidase, is shown in Fig. 2. Under these conditions the reaction was better sustained than under the conditions used in Fig. 1, in which H202 was added as a bolus. The reaction in Fig. 2 was carried out at pH 7.0, close to the optimal pH for glucose oxidase. As in the case of the 1-min incubation at pH 5.4 shown
separate experiments are shown in Table I. The incubation system was the same as that used in Fig. 2, except that the I- concentration was 50 yM instead of 100 PM. The Tg was digested and analyzed after 45 min of incubation. For comparison, results are also shown for Tg iodinated under the same conditions with TPO. The results in Table I demonstrate that MPO, in the presence of 0.1 N Cl-, is almost as active as TPO in catalyzing the formation of T4 and T3. These results suggested that MPO catalyzed coupling as well as iodination. Evidence for this is presented in the following section. Coupling activity of MPO compared to TPO. As described more fully under Methods and Materials, coupling activity was measured by the increase in radiolabeled T, and T3 obtained when 12511or 1311-Tgcontaining the radiolabel primarily as DIT and MIT was further incubated
70 @J
25nMMPOorTPO 100 &4 ‘2% 1 CM g&f Tg 1mghnlW 0.5 llgm ghliwM oxklaw 67 mM phosphate. pH 7.0 37”
80 :
?IJ 50 x i
40
30
10
0
20
10 Incubation
30
Time (min)
FIG. 2. MPO- and TPO-catalyzed iodination of goiter Tg at pH 7 with H,Oz generated by glucose-glucose oxidase; effect of 0.1 N Cl-. The reaction was initiated by addition of glucose oxidase and aliquots were removed at 10 and 20 min and added to a small volume of 0.5 M methimazole solution to stop the reaction. At 30 min a small volume of 0.5 M methimazole was added to the remaining incubation solution. Proteinbound iodine was separated from unreacted iodide by paper chromatography.
242
TAUROG AND DORRIS TABLE
T4, T3, DIT, and MIT
Expt. 1 2
Formation
Enzyme
cl-
I atoms per molecule ‘&
TPO MPO TPO MPO MPO
0 0.1 N 0 0 0.1 N
48.4 49.5 45.2 30.4 46.6
in Goiter
I
Thyroglobulin
% of radioiodine
Iodinated
with
MPO
in pronase digest
or TPO Residues per molecule Tg
T4
T3
DIT
MIT
T4
T3
DIT
18 17 18 15 15
0.97 0.89 0.96 2.2 0.79
39 43 37 31 41
28 27 27 36 27
2.2 2.1 2.1 1.1 1.8
0.16 0.15 0.14 0.23 0.12
9.5 11 a.4 4.7 9.6
Note. The incubation mixture contained 1 pM goiter Tg, 50 pM i3iI- (Experiment 1) or 50 pM is61- (Experiment 1 mg/ml glucose, and 0.5 pg/ml glucose oxidase, in 67 mM phosphate buffer, pH 7.0.
MIT 13 13 12 11 13
2), 26 nM MPO or 27 nM TPO,
with peroxidase + glucose-glucose oxidase. Results of one curve in the absence of Cl- or Br- was similar to that observed in Fig. 1, except that BSA was more readily ioof several such experiments are shown in Table II. The prelabeled Tg contained only 1.5% of its radioio- dinated than goiter Tg. The more rapid iodination of BSA dine as Tq. On incubation with MPO + glucose-glucose could not be attributed to the higher H202 concentration, oxidase this value rose to 11%. The comparable value for since we observed in a preliminary experiment that MPOTPO coupling was only 5.4% (considerably lower than catalyzed iodination of BSA gave practically identical rethe 7-9% we generally obtain with this enzyme). Thus, sults with 100 and 300 PM H202 over the pH range 3.5 under the conditions of our coupling experiments, MPO 6.5. It is of interest that Br-, like Cl-, greatly inhibited was more efficient than TPO in catalyzing the biosyn- MPO-catalyzed iodination at low pH. thesis of Tq. Formation of T3 was much less than that of Nonenzymatic iodination of BSA and goiter Tg with Tq, but in this reaction also, MPO appeared to be some- HOCZ. It is well known that MPO catalyzes the oxidawhat more active than TPO. tion of Cl- to HOC1 at pH 5 (30-32). The HOC1 formed As shown previously (21,27-29), the coupling reaction in this reaction is theoretically capable of oxidizing I- to with TPO and with LPO is greatly enhanced in the pres- an iodinating species. To demonstrate that this actually ence of 1 PM DIT. MPO-catalyzed coupling was also enhanced by 1 PM DIT, as shown in Table II. Although TABLE II coupling by MPO in the absence of added DIT exceeded that by TPO, DIT-stimulated coupling appeared to be Coupling Activity of MPO and TPO somewhat greater with TPO than with MPO. The presence of 0.1 N Cl- had an inhibitory effect on Labeled iodoamino acid distribution after coupling % DIT-stimulated coupling with MPO (Table II) in contrast of radioiodine in pronase digest to its stimulatory effect on MPO-catalyzed iodination Enzyme used clDIT (Table I, Fig. 2). However, 0.1 N Cl- had no effect on DITDIT MIT for coupling (mM) (PM) Td Ts stimulated TPO-catalyzed coupling, and it had no effect on MPO-catalyzed coupling in the absence of DIT (Table 0 0 11 2.1 35 40 MPO II). At present, we have no explanation for the inhibitory 1 0 16 3.5 27 39 MPO 0 11 1.6 38 40 100 MPO effect of 0.1 N Cl- on DIT-stimulated coupling with MPO. 1 12 1.7 36 40 MPO 100 Table III shows the results of an experiment in which 0 0 5.4 1.1 43 40 TPO the coupling activity of MPO was compared to that of 1 25 38 0 18 4.5 TPO TPO at varying enzyme concentrations. Results for both 1 17 4.2 25 38 TPO 100 bovine and human MPO are presented. Only DIT-stimNote. The labeled Tg was prepared by nonenzymatic iodination of ulated coupling was measured. Coupling increased only slightly with increasing concentration of enzyme, and the goiter Tg with ‘*‘I;, as described in the Materials and Methods section. The initial labeled Tg contained 23.5 atoms I/molecule and the ‘*‘Iactivity of both h-MPO and b-MPO compared favorably labeled iodoamino acid distribution was 1.5% Th, 0.25% Ts, 52% DIT, with that of TPO at all concentrations. and 40% MIT. The initial labeled iodoamino acid distribution was deComparison of effect of Br- and Cl- on MPO-catalyzed iodination. As shown in Fig. 3,0.1 N Br- was even more
effective than 0.1 N Cl- in stimulating MPO-catalyzed iodination. The iodine acceptor in this experiment was BSA, and the HzOz concentration was 300 PM. The pH
termined after pronase digestion of labeled Tg incubated with glucoseglucose oxidase alone. The coupling incubation system contained 1.5 gM labeled Tg, 25 nM MPO or TPO, 0 or 100 mM NaCl, 0 or 1 pM DIT, 1 mg/ml glucose, and 0.1 rg/ml glucose oxidase, in 65 mM phosphate buffer, pH 7.0. The reaction was initiated with glucose oxidase, and samples were incubated for 20 min at 37°C.
MYELOPEROXIDASE-CATALYZED
IODINATION
occurs, we tested iodination of BSA and goiter Tg with NaOCl at pH 5.4 (Fig. 4). At this pH OCll exists almost entirely as HOC1 (p& = 7.5). In an incubation mixture containing 7.5 PM BSA and 100 PM lz51-, addition of 300 FM NaOCl produced binding of 77% of the 1251in 1 min. With 1 PM goiter Tg as the acceptor, 46% of the 1251was similarly bound. Iodination occurred even more readily under these conditions than in the MPO-catalyzed reactions of Fig. 1 and Fig. 3. As in the MPO-catalyzed reaction, BSA was more readily iodinated than goiter Tg. These results provide a possible explanation for the stimulatory effect of Cl- on MPO-catalyzed iodination of protein at pH 5.4, observed in Figs. 1 and 3. Effectof thioureylene drugs on MPO-catalyzed i&nation of BSA. The thioureylene antithyroid drugs, B-propylthiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MMI, methimazole) are well known inhibitors of TPOcatalyzed iodination. Figure 5 shows the effect of varying concentrations of PTU and MM1 on MPO-catalyzed iodination of BSA. The results with MPO show some of the same features that we have previously reported (33, 34) for TPO-catalyzed iodination (see Discussion section). DISCUSSION TPO was the first animal peroxidase whose primary amino acid sequence was deduced from its nucleotide sequence (3-6). Shortly thereafter, the primary amino acid
TABLE Effect
of Enzyme with
III
Concentration on Coupling MPO and TPO Labeled iodoamino acid distribution after coupling % of i3iI in pronase digest
Enzyme h-MPO
b-MPO
TPO
Enzyme concn. (nM)
T4
T3
DIT
MIT
5 12.5 25 5 12.5 25 5 12.5 25
11 13 14 12 13 14 13 15 16
1.5 2.1 2.6 1.8 2.5 3.2 2.1 3.4 3.8
37 33 30 34 31 28 34 28 26
43 43 43 43 42 42 42 41 40
Note. The labeled Tg was prepared by nonenzymatic iodination of goiter Tg with r3*I;, as described in the Methods and Materials section. The I’iI-Tg initially contained 26.3 atoms I/molecule and the following i3iI distribution: Tp, 1.8%; Ts, 0.24%; DIT, 50%; MIT, 42%. The coupling incubation system contained 1.44 j&M labeled Tg, 1 PM DIT, 1 mg/ml glucose, and 0.1 fig/ml glucose oxidase, in 65 mM phosphate buffer, pH 7.0. The reaction was initiated with glucose oxidase, and all samples were incubated for 20 min at 37°C. Each value is the mean of closely agreeing duplicates.
AND
243
COUPLING
so 70 t
u 602 ‘E 50e 0
40-
!
30-
3 20 IOOfI
I
1
I
I
1
I
I
1
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
FIG. 3. MPO-catalyzed iodination of BSA at various pHs; effect of 0.1 N Cl- and 0.1 N Br-. See legend to Fig. 1. The effect of 0.1 N Br- on pH was very similar to that of 0.1 N Cl-.
sequence of MPO was determined in the same manner (7, 8), and more recently that of LPO (9). Before the sequence of LPO was published, Kimura and Ikeda-Saito (35) compared the sequences of TPO and MPO and concluded that, even though the two enzymes have distinctly different physiological functions, they are evolutionarily related members of the same gene family. The more recent data with LPO suggests that it too is a member of the same gene family. Both MPO and LPO show significant cross reactivity with autoantibodies to TPO, which are frequently found in serum from patients with autoimmune thyroid disease (36). It was early demonstrated that purified MPO catalyzes iodination of tyrosine (ll), BSA (12), and thyroglobulin.4 It has also been shown (13) that MPO, covalently bound to Sepharose, can be used to iodinate a variety of proteins. MPO is a major component of the oxygen-dependent bactericidal system in leucocytes, and its ability to catalyze iodination forms the basis of a screening test for cellular or humoral abnormalities in the phagocytic process (14). In the present study we focused primarily on MPOcatalyzed iodination of Tg and on the ability of MPO to catalyze the formation of thyroxine. Aside from a preliminary observation reported many years ago,4 it was not known whether MPO, like TPO and LPO, catalyzes the coupling reaction. Iodination and coupling are very different types of chemical reaction and almost certainly involve different reaction mechanisms. In this connection, ’ Taurog, A., and Gamble, D. E. (1966) Fed. Proc. 26.347.
[Abstract]
244
TAUROG
90
1
SOI
;
50
AND
BSA
/
r-O
goiterTg
40
1oopM ‘2513OOpM NaOCl
30
67 mM phosphatebuffer, pH
5.4
room temperature
Ilr
“y, ( ( , , 1
2
3
4
5
incubation Time (min) FIG. 4. Nonenzymatic iodination of BSA and goiter Tg with NaOCl. The incubation mixture contained either 7.5 pM BSA or 1 pM goiter Tg. Other conditions were as indicated in the figure. The reaction was initiated with NaOCl and stopped after, 1,3, or 5 min by the addition of sodium thiosulfate (final concentration, 10 mM). Protein-bound iodine was separated from unreacted iodide by paper chromatography.
it is of interest that cytochrome c peroxidase readily catalyzes the coupling reaction, but shows no activity in the iodination of Tg or BSA [(37), Taurog and Dorris, unpublished observations]. We were unable to find in the literature a systematic study of the effect of pH on MPO-catalyzed iodination of protein. Also, in preliminary experiments reported many years ago (12), we observed that 0.1 N Cl- had a marked stimulatory effect on MPO-catalyzed iodination of BSA. In our present study, therefore, involving MPO-catalyzed iodination of goiter Tg or BSA, we first examined the effects of pH and 0.1 N Cl-. Iodination was measured 1 min after the reaction was initiated with H202 to approximate initial rates of iodination. Comparable studies were performed with TPO and LPO. MPO-catalyzed iodination of Tg was markedly affected by pH and by 0.1 N Cl- or Br-. In the absence of Cl- or Br- the pH optimum for iodination of Tg was about 4.0, much lower than that observed for LPO (5.4) or for TPO (6.6). In the presence of 0.1 N Cl-, the pH optimum for MPO-catalyzed iodination shifted to about 5.5. At this pH 0.1 N Cl- markedly stimulated the rate of iodination. At pH 4.0, the optimum pH in the absence of Cl-, the addition of 0.1 N Cl- inhibited iodination by more than 98%. This is consistent with the model proposed by An-
DORRIS
drews and Krinsky (26), suggesting two distinct Cl- binding sites on MPO, one acting as a substrate site for oxidation of Cl- to HOC1 and the other as an inhibitory site. According to their model, the latter site requires prior protonation and has an estimated pK, of about 4.5. Our observation that MPO-catalyzed iodination is markedly inhibited by 0.1 N Br- at pH 4.0 (Fig. 3) suggests that the Cl- inhibitory site is also sensitive to excess Br-. We attempted to compare Km values for MPO- and TPO-catalyzed iodination of Tg and BSA by measuring initial rates of iodination at varying concentrations of iodide. However, in the case of MPO we observed a lag phase in the kinetics of iodination, in both the presence and absence of Cl-. Such a lag was not observed with TPO or LPO. We were unable, therefore, to obtain reliable measurements of initial rates of MPO-catalyzed iodination. At present, we have no adequate explanation for this lag phase and until this problem is resolved, we are unable to determine a meaningful K,,, value for MPOcatalyzed iodination. The marked stimulatory and inhibitory effects of Clon MPO-catalyzed iodination were not observed with TPO or with LPO. A major difference between MPO and TPO or LPO is that MPO is capable of oxidizing Cl- to HOCl. The pH optimum of this reaction is about 5.5 (31), very close to the pH optimum of Cl--stimulated, MPOcatalyzed iodination of goiter Tg and BSA observed in the present study (Figs. 1 and 3). These results suggested that the stimulatory effect of Cl- involves oxidation of Iby HOC1 to an iodinating species (e.g., HOI) that can iodinate tyrosyl residues of protein nonenzymatically. Evidence for this was obtained by our demonstration (Fig. 4) that BSA and goiter Tg are readily iodinated by a mixture of NaOCl and I- at pH 5.4 in the absence of peroxidase or HzOz. At this pH OCll exists primarily as HOCl.
Glucose 1 mgml Glumse Oxidase 0.5 tin-d
T) s 8
80
8o
%
40
10
20
30
40
80
80 Incubation
Time (min)
FIG. 5. Effect of antithyroid drugs, MM1 and PTU, on MPO-catalyzed iodination of BSA. b-MPO was used in this experiment. Similar results were obtained with h-MPO.
MYELOPEROXIDASE-CATALYZED
We have previously suggested (38) that the function of TPO and LPO in iodination of tyrosine and tyrosyl residues in protein may be to generate HOI, which could then iodinate nonenzymatically. A similar proposal was made for HRP-catalyzed iodination of tyrosine (39). In a study performed many years ago (40) with chloroperoxidase (CPO), we observed that CPO-catalyzed iodination of tyrosine and thyroglobulin was even more markedly stimulated by 0.1 N Cl- than was MPO-catalyzed iodination in the present study. We suggested that an oxidized form of Cl- was an intermediate in oxidizing I- to an iodinating species. It now appears likely that HOC1 was the intermediate involved in our previously observed stimulatory effect of Cl- on CPO-catalyzed iodination. For studying T4 formation in the MPO-catalyzed iodination of Tg, we used an incubation system at pH 7.0 containing the HzOz generating system, glucose-glucose oxidase (Fig. 2), and an incubation period of 45 min. The iodinating activity under these conditions was also stimulated by 0.1 N Cl-. The mechanism of this stimulatory effect may also be mediated through the formation of HOCl/OCll. We have shown previously (38) that the steady state level of HzOz during TPO-catalyzed iodination in the presence of glucose-glucose oxidase is very low, especially when compared with the 100 PM H202 added for the 1-min incubations in Fig. 1. Thus, the Cl-/ H202 ratio was much greater in the system containing glucose-glucose oxidase (Fig. 2) than it was under the conditions used in Fig. 1. As shown by previous investigators (31, 41), this would shift the pH optimum of Cloxidation to more alkaline pHs. It is likely, therefore, that under the conditions used in Fig. 2 (pH 7.0, glucose-glucose oxidase), there was significant oxidation of Cl- to HOCl/OCll by MPO. There was also very significant formation of T*, comparable to that observed in a similar incubation system containing TPO, with or without Cl-. MPO was also tested in a coupling system designed to measure T4 formation in the absence of iodination (Tables 2 and 3). In the absence of free DIT it was more active than TPO on a molar basis under the conditions used in Table II. However, in the presence of a low concentration of free DIT, which acts to stimulate the coupling reaction, TPO was more active than MPO. We have previously proposed a radical mechanism for the TPO-catalyzed coupling reaction (l), based on suggestions of previous investigators (43,44). The same mechanism may also apply to MPO-catalyzed coupling. Peroxidase-catalyzed iodination, on the other hand, is generally regarded as a two-electron reaction (l), in which I- is oxidized directly by Compound I to I+ or its equivalent oxidation state, HOI. MPO-catalyzed iodination was inhibited by the thioureylene antithyroid drugs, MM1 and PTU, in a manner similar to that previously observed for TPO-catalyzed iodination (33, 34). At lower drug concentrations (Fig. 5),
IODINATION
AND
245
COUPLING
inhibition of iodination was transient, and after a variable interval, dependent on the drug concentration, there was escape from inhibition. In the case of TPO, we have shown that the period of inhibition is associated with extensive, iodide-dependent, drug oxidation. At higher drug concentrations, however, the enzyme is rapidly inactivated by the drug, and only limited drug oxidation occurs before the enzyme is inactivated. We have previously (34) proposed a scheme to explain reversible and irreversible inhibition of TPO-catalyzed iodination by MM1 and PTU. Although only limited data were obtained for inhibition of MPO-catalyzed iodination by MM1 and PTU, the results in Fig. 5 suggest that the same general scheme may also apply to MPO-catalyzed iodination. REFERENCES 1. Taurog, A. (1991) in Werner’s The Thyroid (Braverman, L. E., and Utiger, R. D., Eds.), 6th ed., pp. 51-97, J. B. Lippincott, Philadelphia. 2. Taurog, A., Dorris, M. L., and Lamas, L. (1974) Endocrinology 1286-1294.
94,
3. Magnusson, R. P., Gestautas, J., Taurog, A., and Rapoport, B. (1987) J. Bid. Chem. 262, 13,885-13,888. 4. Kimura, S., kayama, T., 5555-555s. 5. Magnusson, S., DeGroot, 861.
Kotani, T., McBride, 0. W., Umeki, K., Hirai, K., Naand Oktaki, S. (1987) Proc. N&l. Acad. Sci. USA 84, R. P., Chazenbalk, G. D., Gestautas, J., Seto, P., Filleti, L. J., and Rapoport, B. (1987) Mol. Endocrinol. 1,856-
6. Libert, F., Ruel, J., Ludgate, M., Swillens, S., Alexander, G., and Dinsart, C., (1987) EMBO J. 6, 4193-4196.
N., Vassart,
7. Johnson, K. R., Nauseef, W. M., Care, A., Weelock, M. J., Shane, S., Hudson, S., Koeffler, H. P., Selsted, M., Miller, C., and Rovera, G. (1987) Nucleic Acids Res. 15, 2013-2028. 8. Morishita, K., Kubota, N., Asano, S., Kaziro, Y., and Nagata, S. (1987) J. Biol. Chem. 262, 3844-3851. 9. Dull, T. J., Uyeda, C., Strosberg, A. D., Nedwin, G., and Seilhamer, J. J. (1990) DNA Cell Biol. 9, 499-509. 10. Banga, J. P., Sutton, B. J., Barton, G. J., Mahadevan, D., Saldanha, J., and McGregor, A. M. (1991) in Progress in Thyroid Research: Proceedings of the 10th International Thyroid Conference (Gordon, A., Gross, J., and Henneman, G., Eds.), pp. 433-436, A. A. Balkeman, Rotterdam. 11. Klebanoff, S. J., Yip, C., and Kessler, D. (1962) Biochim. Biophys. Actu 58, 563-574. 12. Coval, M. L., and Taurog, A. (1967) J. Biol. Chem. 242,5510-5523. 13. Dubin, A., and Silberring, 14. Klebanoff, 686.
S. J., andclark,
J. (1976) Anal. Biochem. 72, 372-379. R. A. (1977) J. Lab. Clin. Med. 89,675-
15. Turk, J., Henderson, W. R., Klebanoff, S. J., and Hubbard, (1983) Biochim. Biophys. Acta 751, 189-200.
W. C.
16. Agner, K. (1958) Acta Chen. Stand. 12,89-94. 17. Marquez, L. A., Dunford, Chem. 265, 5666-5670.
H. B., and Van Wart, H. (1990) J. Biol.
18. Morrison, M., and Bayse, G. S. (1970) Biochemistry 9,2995-3000. 19. Rawitch, A. B., Taurog, A., Chernoff, S. B., and Dorris, M. L. (1979) Arch. Biochem. Biophys. 194, 244-257. 20. Yokoyama, N., and Taurog, A. (1988) Mol. Endocrinol. 2,838-844. 21. Taurog, A., and Nakashima, T. (1978) Endocrinolog.y 103,632-640.
246
TAUROG
22. Salvatore, G., Vecchio, G., Salvatore, M., Cahnmann, Robbins, J. (1965) J. Biol. Chem. 240, 2935-2943.
AND
H. J., and
23. Jencks, W. P., and Regenstein, J. (1976) in Handbook of Biochemistry and Molecular Biology (Fasman, G. D., Ed.), 3rd ed., Vol. 1, pp. 305-384, CRC Press Inc., Cleveland. 24. Lamas, L., Dorris, M. L., and Taurog, A. (1972) Endocrinology 1417-1426.
90,
25. Taurog, A., Riesco, G., and Larsen, P. R. (1976) Endocrinology 281-290.
99,
26. Andrews, P. C., andKrinsky, 13,245.
N. I. (1982) J. Biol. Chem. 257,13,240-
27. Deme, D., Fimiani, J., Pommier, Biochem. 51,329-336.
J., and Nunez, J. (1975) Eur. J.
DORRIS
33. Engler, H., Taurog, A., Luthy, crinology 112,86-95.
C., and Dorris,
M. L. (1983) Endo-
34. Taurog, A., Dorris, M. L., and Gusiec, F. S., Jr. (1989) Endocrinology
124,30-39. 35. Kimura, S., and Ikeda-Saito, M. (1988) Proteins: Struct. Funct. &net. 3,113-120. 36. Banga, J. P., Tomlinson, R. W. S., Doble, N., Odell, E., and McGregor, A. M. (1989) Immunology 67,197-204. 37. Deme, D., Virion, A., Michot, J., and Pommier, J. (1985) Arch. Biochem. Biophys. 236, 559-566. 38. Magnusson, R., Taurog, A., andDorris, M. L. (1984) J. Biol. Chem. 259, 13,783-13,790.
28. Courtin, F., Deme, D., Virion, A., Michot, J. L., Pommier, Nunez, J. (1982) Eur. J. Biochem. 124, 603-609.
J., and
29. Virion, A., Courtin, F., Deme, D., Michot, J. L., Kaniewski, Pommier, J. (1985) Arch. Biochem. Biaphys. 242, 41-47.
J., and
39. Dunford, H. B., and Ralston, I. M. (1983) Biochem. Biophys. Res. Commun. 116,639-643. 40. Taurog, A., and Howells, E. M. (1966) J. Biol. Chem. 241, 13291339.
J. E., and Schultz, J. (1976) J. Biol. Chem. 251, 1371-
41. Zgliczynski, J. M., Selvaraj, R. J., Paul, B. B., Stelmaszynska, T., Poskit, P. K. F., and Sbarra, A. J. (1977) Proc. Sot. Exp. Biol. Med.
30. Harrison, 1374.
31. Bakkenist, A. R. J., deBoer, J. E. G., Plat, H., and Wever, R. (1980) Biochim. Biophys. Acta 613, 337-348. 32. Kettle, A. J., and Winterbourn, 957,185-191.
C. C. (1988) Biochim. Biophys. Acta
154,418-422. 42. Taurog, A. (1970) Recent Prog. Hormone Res. 26, 189-247. 43. Johnson, T. B., and Tewkesbury, USA 26, 73-77. 44. Harington,
L. B. (1942) Proc. Natl. Acad. Sci.
C. R. (1944) J. Chem. Sot., 193-201.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 296, No. 1, July, pp. 239-246, 1992
Myeloperoxidase-Catalyzed Alvin Taurog2 and Martha Department
of Pharmacology,
Received November
lodination
L. Dorris
University
of Texas Southwestern
Press, Inc.
‘ This work was supported by research grants from Boots Pharmaceuticals and from the United States Public Health Service (NIDDK03612). ’ TO whom correspondence should be addressed.
Thyroid peroxidase (TP0)3, an enzyme that plays an essential role in the formation of thyroid hormones, catalyzes both iodination of thyroglobulin and the intramolecular coupling of two diiodotyrosine (DIT) molecules to form thyroxine (T4) and of one monoiodotyrosine (MIT) molecule and one DIT molecule to form 3’,3,5triiodothyronine (T3) (1). We previously observed (2) that lactoperoxidase (LPO), an enzyme isolated from milk, also catalyzes these reactions with much the same efficiency as TPO. Both purified TPO and LPO have been employed in model systems to study mechanisms of peroxidase-catalyzed iodination and coupling (1). More recently, the complete primary amino acid sequences of porcine (3) and human (4-6) TPO, human myeloperoxidase (MPO) (7,8), and bovine LPO (9) have been deduced from cDNA cloning experiments. These sequences display considerable homology, especially in regions considered to be important for binding the heme prosthetic group (10). Although myeloperoxidase (MPO) is known to catalyze iodination reactions (11-S), very little has been reported regarding its ability to catalyze iodination of thyroglobulin (Tg). The sequence similarities with TPO and LPO, together with our previous studies demonstrating that LPO as well as TPO catalyzes the coupling reaction, suggestedthat MPO also might catalyze T4 biosynthesis in Tg. It was the purpose of this study to investigate this possibility, and also to examine the effect of pH and of chloride on MPO-catalyzed iodination of Tg. METHODS
AND
MATERIALS
Human MPO was purchased from Alpha TheraMyeloperoxidase. peutic Corp. (Los Angeles, CA). The lyophilized product, containing a high concentration of salt, was dialyzed against 67 mM phosphate buffer, pH 7.0, and aliquots of the frozen solution were stored at -20°C. The value for Aao/Am was 0.74, and enzyme concentration was calculated
3 Abbreviations used: MPO, myeloperoxiclase; TPO, thyroid peroxidase; LPO, lactoperoxidase; HRP, horseradish peroxidase; Tg, thyroglobulin; T*, thyroxine; Ts, 3’,3,5-triiodothyronine; DIT, diiodotyrosine; MIT, monoiodotyrosine; MMI, 1-methyl-2-mercaptoimidazole; PTU, 6-propyl-2-thiouracih BSA, bovine serum albumin.
239
$5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction
Medical Center, Dallas, Texas 75235
8,1991, and in revised form March lo,1992
Myeloperoxidase (MPO), which displays considerable amino acid sequence homology with thyroid peroxidase (TPO) and lactoperoxidase (LPO), was tested for its ability to catalyze iodination of thyroglobulin and coupling of two diiodotyrosyl residues within thyroglobulin to form thyroxine. After 1 min of incubation in a system containing goiter thyroglobulin, II, and HzOz, the pH optimum of MPO-catalyzed iodination was markedly acidic (-4.0), compared to LPO (-5.4) and TPO (-6.6). The presence of 0.1 N Cl- or Br- shifted the pH optimum for MPO to about 5.4 but had little or no effect on TPO- or LPO-catalyzed iodination. At pH 5.4, 0.1 N Cl- and 0.1 N Br- had a marked stimulatory effect on MPO-catalyzed iodination. At pH 4.0, however, iodinating activity of MPO was almost completely inhibited by 0.1 N Cl- or Br-. Inhibition of chlorinating activity of MPO by Cl- at pH 4.0 has been previously described. When iodination of goiter thyroglobulin was performed with MPO plus the HzOz generating system, glucose-glucose oxidase, at pH 7.0, the iodinating activity was markedly increased iodination and thyby 0.1 N Cl-. Under these conditions roxine formation were comparable to values observed with TPO. MPO and TPO were also compared for coupling activity in a system that measures coupling of diiodotyrosyl residues in thyroglobulin in the absence of iodination. MPO displayed very significant coupling activity, and, like TPO, this activity was stimulated by a low concentration of free diiodotyrosine (1 FM). The thioureylene drugs, propylthiouracil and methimazole, inhibited MPO-catalyzed iodination both reversibly and irreversibly, in a manner similar to that previously described for TPO-catalyzed iodination. o 1992 Academic
0003-9861/92
and Coupling’
in any form reserved.
240
TAUROG
AND
on the basis of a millimolar extinction coefficient at 430 nm of 178 (16, 17). Some experiments were performed with bovine MPO, purchased from ExOxEmis (San Antonio, TX), after human MPO became unavailable from our original supplier. The bovine MPO displayed a value of 0.72 for A,,O/Azso, and based on our guaiacol assay it was about 17% more active (per mole of heme) than the human MPO. Lactoperoxidase. Bovine LPO was obtained from Pharmacia or Sigma. It was dialyzed against 67 mu phosphate, pH 7.0, and the enzyme concentration was based on a millimolar extinction coefficient at 412 nm of 114 (18). The value for A412/A280was 0.89-0.90. Thyroid peroxidase. Porcine TPO (Preparation XIII) was prepared in this laboratory as previously described (19, 20). It is a highly active, trypsin-detergent-solubilized product, whose relationship to the native enzyme has been characterized (20). The value for Ad12/Am was 0.48. The extinction coefficient for TPO has not been accurately determined, and in this study it was assumed to be the same as that for LPO. Goiter thyroglobulin. Thyroglobulin (Tg) was isolated from a large goitrous human thyroid and purified by repeated gel filtration through Bio-Gel A-5m (21). The final lyophilized material contained 0.038% I, equivalent to an average of two atoms of I per molecule. The concentration of Tg was determined by measurement of A*, using an extinction coefficient of 10.0 for a 1% solution (22). Reagents. BSA (crystallized) was obtained from ICN Biochemicals, glucose oxidase (lyophilized, 100 U/mg) from Boehringer Mannheim, PTU and MM1 from Aldrich Chemical Co., and NaOCl (analytical reagent) from Mallinckrodt Chemical CO. Zodinution procedure. The incubation mixture contained goiter Tg or BSA, peroxidase, radiolabeled iodide, and either directly added HzOz or the H202 generating system, glucose-glucose oxidase. The concentrations of the various components are provided with the results of individual experiments. In experiments designed to study the effect of pH, the pH was varied from 3.5 to 7.5. Phosphate buffer (67 mM) was used for pH 5.4 and above, and acetate buffer (0.1 M) for pHs below 5.4. The incubation mixture in many cases contained 0.1 N NaCl, and in some cases 0.1 N NaBr. We observed that the presence of 0.1 N Cl- or 0.1 N Br- in phosphate buffers decreased the measured pH by 0.1-0.2 pH units. A decrease was also observed with acetate buffer, but of smaller magnitude. These results are consistent with the known effect of ionic strength on the pK,, of neutral and anionic acids (23). Measured pH values are shown in the figures presented in the Results section. Experiments with added HzOz were used to approximate initial rates of iodination. The reaction was initiated with H,Oz and stopped after 1 min by addition of a large excess of I-methyl-2-mercaptoimidazole (MMI, methimazole, final concentration, 5 mM). Protein-bound iodine was separated from unreacted iodide by paper chromatography in collidine-NH,OH, as previously described (24). Results are expressed as pM I- bound to protein in 1 min. For measurements of T4 formation in Tg iodinated with MPO or TPO, the iodination was performed with H,Os generated by glucoseglucose oxidase. Under these conditions the degree of iodination increased steadily for at least 30 min. The reaction was initiated with glucose oxidase and stopped with methimazole after a 45-min incubation at 37°C. The Tg was digested with pronase plus aminopeptidase, and labeled T1, Ts. MIT, and DIT were separated by paper chromatography, as previously described (25). The corresponding sections of the chromatogram, visualized by radioautography, were counted, and residues of T4, T3, MIT, and DIT were calculated from the degree of iodination and the radioiodine distribution. Coupling with chemically labeled Tg. Goiter Tg was iodinated chemically with radiolabeled 1, to a level of 23-29 atoms I per molecule. The radiolabeled 1; solution contained 7.07 mM IZ (determined by titration with standard thiosulfate) in 26 mM Kl and a very small volume of iz61
DORRIS
or i3iI, added to yield a specific activity of about 100 or 200 pCi/geq I, respectively. The labeled I; was added to 5-10 ml of a magnetically stirred solution of 1.5 ELMTg (7 ~1 of I; per ml of Tg), and after 2 min of stirring, 10 ~1 of 1 M sodium thiosulfate was added to reduce unreacted I;. The number of iodine atoms bound per molecule of Tg was calculated from the amount of radioiodine remaining at the origin of the paper chromatogram (protein-bound), the specific activity of the iodine in the labeled 1, solution, and the protein concentration. The labeled Tg solution was dialyzed thoroughly against 67 mM phosphate pH 7.0 at 4°C (400 vol, two changes of buffer) over a period of 22 h to remove inorganic radioiodine. After dialysis, the labeled Tg, which contained almost all of its radioiodine as DIT and MIT, and very little as Tq, was incubated with MPO or TPO under the following conditions: 1.5 pM Tg, 25 nM MPO or TPO, 1 mg/ml glucose, and 0.1 pg/ml glucose oxidase, in 65 mM phosphate, pH 7.0, for 20 min at 37°C. The reaction was stopped with excess methimazole, and the incubation mixture was digested with pronase plus aminopeptidase and the labeled iodoamino acid distribution determined as previously described (25). Coupling activity was measured by the increase in the percentage of radioiodine present as T4 + Ts, compared to the percentages in labeled Tg incubated in the same system lacking peroxidase. Zodination of BSA andgoiter Tg with NaOClplus Z-. Results obtained in the present study with MPO-catalyzed iodination at pH 5.4 in the presence of 0.1 N Cl- suggested that HOC1 might be an intermediate in the iodination reaction. To test this possibility we used a solution of NaOCl for iodination of BSA and goiter Tg. The NaOCl was standardized by iodimetric titration. The incubation mixture contained either 7.5 PM BSA or 1 pM goiter Tg, 100 PM izr’I-, and 300 PM NaOCl, in phosphate buffer, pH 5.4. The reaction was initiated with the NaOCl and was terminated after 1, 3, or 5 min by the addition of sodium thiosulfate (final concentration, 10 mM). Protein-bound ‘%I was determined by paper chromatography, as described above. Inhibition of MPO-catalyzed iodination by 6-propylthiouracil (PTU) and I-methyl-2-mercaptoimidazole (MMZ). The incubation system contained 25 nM bovine MPO, 7.5 PM BSA, 100 pM i311-, 0.1 N NaCl, 1 mg/ml glucose, and 0.5 pg/ml glucose oxidase, in 60 mM phosphate buffer, pH 7.0. Drug concentrations varied from 0 to 300 FM. After initiation of the reaction at 37’C with glucose oxidase, samples were removed at 3,10,25,45, and 65 min and added to small tubes containing a small volume of 0.5 M MM1 to stop the reaction. The tubes were placed in an ice bath at O”C, and 25 ~1 of the reaction mixture was applied for filter paper chromatography in co&dine-NH,OH to determine the fraction of the 1311bound to protein.
RESULTS Effect of pH and Cl- on MPO-, TPO-, and LPO-catalyzed iodination of goiter Tg. Figure 1 shows results obtained after 1 min of incubation in a system containing MPO, TPO, or LPO, goiter Tg, labeled iodide, and H202 at varying pH and in the presence and absence of 0.1 N Cl-. Results are expressed as PM I- bound in 1 min. MPO readily catalyzed iodination of Tg in a pH- and Cl-dependent manner. In the absence of Cl-, the pH optimum of the reaction was about 4.0, and under the conditions of the incubation 2.3 X lo3 mol of I- was bound in 1 min per mole of enzyme. In the presence of 0.1 N Cl-, the pH optimum shifted to about 5.4, and 2.1 X lo3 mol of I- was bound in 1 min per mole of enzyme. At pH 4.0, 0.1 N Cl- was a potent inhibitor of iodination. This may be attributed to an inhibitory binding site for Cl- on
MYELOPEROXIDASE-CATALYZED
2 28 .E E t .E 24 z 3
20-
i
16-
100 ;M H202 or 0.1 N Cl-
I0
IODINATION
AND
241
COUPLING
in Fig. 1, iodinating activity catalyzed by MPO was significantly stimulated in the presence of 0.1 N Cl-. After a 30-min incubation, 79% of the I- was bound to Tg, compared to 50% in the absence of Cl-. When 0.1 N Cl- was present in the incubation mixture, MPO catalyzed the iodination of Tg as readily as did TPO (Fig. 2). However, in contrast to its effect on MPO-catalyzed iodination, Clhad no effect on TPO-catalyzed iodination (Fig. 2), or on LPO-catalyzed iodination (results not shown).
I
Iodoamino acid distribution in Tg iodinated with the MPO-glucose-glucose oxidase system. Results of two
12a-
1
5.0
I
5.5 PH
I
I
1
I
6.0
6.5
7.0
7.5
FIG. 1. Peroxidase-catalyzed iodination of goiter Tg at various pHs with and without 0.1 N Cl-; comparison of MPO, TPO, and LPO. The reaction was initiated at 37°C by addition of Hz02 and stopped after 1 min by addition of methimazole (final concn. 5 mM). Phosphate buffer (67 mM) was used for pH 5.4 and above and acetate buffer (0.1 N) for decreased pH pHs below pH 5.4. Addition of 0.1 N Cl- significantly values, especially in phosphate buffers.
MPO, which, as previously described (26), requires prior protonation. Iodination of goiter Tg catalyzed by TPO and by LPO under exactly the same conditions used for MPO was also highly pH-dependent but showed much less effect of Cl-. The pH optimum for TPO was about 6.6, and this was unaffected by 0.1 N Cl-. The peak activity of TPO was 2.7 X lo3 mol of I- bound per mole of enzyme, and this was slightly reduced in the presence of 0.1 N Cl-. LPO displayed maximum activity at about pH 5.4, and under the conditions used it was the most active of the peroxidases, binding 4.3 X lo3 mol of I- per mole of enzyme in one min. The pH optimum and the activity of LPO were little affected by 0.1 N Cl-. MPO-catalyzed iodination of goiter Tg at pH 7.0 with Hz02 generated by glucose-glucose oxidase; effect of 0.1 N
Cl-. The time course of MPO-catalyzed iodination of Tg in an incubation system containing the HzOz generating system, glucose-glucose oxidase, is shown in Fig. 2. Under these conditions the reaction was better sustained than under the conditions used in Fig. 1, in which H202 was added as a bolus. The reaction in Fig. 2 was carried out at pH 7.0, close to the optimal pH for glucose oxidase. As in the case of the 1-min incubation at pH 5.4 shown
separate experiments are shown in Table I. The incubation system was the same as that used in Fig. 2, except that the I- concentration was 50 yM instead of 100 PM. The Tg was digested and analyzed after 45 min of incubation. For comparison, results are also shown for Tg iodinated under the same conditions with TPO. The results in Table I demonstrate that MPO, in the presence of 0.1 N Cl-, is almost as active as TPO in catalyzing the formation of T4 and T3. These results suggested that MPO catalyzed coupling as well as iodination. Evidence for this is presented in the following section. Coupling activity of MPO compared to TPO. As described more fully under Methods and Materials, coupling activity was measured by the increase in radiolabeled T, and T3 obtained when 12511or 1311-Tgcontaining the radiolabel primarily as DIT and MIT was further incubated
70 @J
25nMMPOorTPO 100 &4 ‘2% 1 CM g&f Tg 1mghnlW 0.5 llgm ghliwM oxklaw 67 mM phosphate. pH 7.0 37”
80 :
?IJ 50 x i
40
30
10
0
20
10 Incubation
30
Time (min)
FIG. 2. MPO- and TPO-catalyzed iodination of goiter Tg at pH 7 with H,Oz generated by glucose-glucose oxidase; effect of 0.1 N Cl-. The reaction was initiated by addition of glucose oxidase and aliquots were removed at 10 and 20 min and added to a small volume of 0.5 M methimazole solution to stop the reaction. At 30 min a small volume of 0.5 M methimazole was added to the remaining incubation solution. Proteinbound iodine was separated from unreacted iodide by paper chromatography.
242
TAUROG AND DORRIS TABLE
T4, T3, DIT, and MIT
Expt. 1 2
Formation
Enzyme
cl-
I atoms per molecule ‘&
TPO MPO TPO MPO MPO
0 0.1 N 0 0 0.1 N
48.4 49.5 45.2 30.4 46.6
in Goiter
I
Thyroglobulin
% of radioiodine
Iodinated
with
MPO
in pronase digest
or TPO Residues per molecule Tg
T4
T3
DIT
MIT
T4
T3
DIT
18 17 18 15 15
0.97 0.89 0.96 2.2 0.79
39 43 37 31 41
28 27 27 36 27
2.2 2.1 2.1 1.1 1.8
0.16 0.15 0.14 0.23 0.12
9.5 11 a.4 4.7 9.6
Note. The incubation mixture contained 1 pM goiter Tg, 50 pM i3iI- (Experiment 1) or 50 pM is61- (Experiment 1 mg/ml glucose, and 0.5 pg/ml glucose oxidase, in 67 mM phosphate buffer, pH 7.0.
MIT 13 13 12 11 13
2), 26 nM MPO or 27 nM TPO,
with peroxidase + glucose-glucose oxidase. Results of one curve in the absence of Cl- or Br- was similar to that observed in Fig. 1, except that BSA was more readily ioof several such experiments are shown in Table II. The prelabeled Tg contained only 1.5% of its radioio- dinated than goiter Tg. The more rapid iodination of BSA dine as Tq. On incubation with MPO + glucose-glucose could not be attributed to the higher H202 concentration, oxidase this value rose to 11%. The comparable value for since we observed in a preliminary experiment that MPOTPO coupling was only 5.4% (considerably lower than catalyzed iodination of BSA gave practically identical rethe 7-9% we generally obtain with this enzyme). Thus, sults with 100 and 300 PM H202 over the pH range 3.5 under the conditions of our coupling experiments, MPO 6.5. It is of interest that Br-, like Cl-, greatly inhibited was more efficient than TPO in catalyzing the biosyn- MPO-catalyzed iodination at low pH. thesis of Tq. Formation of T3 was much less than that of Nonenzymatic iodination of BSA and goiter Tg with Tq, but in this reaction also, MPO appeared to be some- HOCZ. It is well known that MPO catalyzes the oxidawhat more active than TPO. tion of Cl- to HOC1 at pH 5 (30-32). The HOC1 formed As shown previously (21,27-29), the coupling reaction in this reaction is theoretically capable of oxidizing I- to with TPO and with LPO is greatly enhanced in the pres- an iodinating species. To demonstrate that this actually ence of 1 PM DIT. MPO-catalyzed coupling was also enhanced by 1 PM DIT, as shown in Table II. Although TABLE II coupling by MPO in the absence of added DIT exceeded that by TPO, DIT-stimulated coupling appeared to be Coupling Activity of MPO and TPO somewhat greater with TPO than with MPO. The presence of 0.1 N Cl- had an inhibitory effect on Labeled iodoamino acid distribution after coupling % DIT-stimulated coupling with MPO (Table II) in contrast of radioiodine in pronase digest to its stimulatory effect on MPO-catalyzed iodination Enzyme used clDIT (Table I, Fig. 2). However, 0.1 N Cl- had no effect on DITDIT MIT for coupling (mM) (PM) Td Ts stimulated TPO-catalyzed coupling, and it had no effect on MPO-catalyzed coupling in the absence of DIT (Table 0 0 11 2.1 35 40 MPO II). At present, we have no explanation for the inhibitory 1 0 16 3.5 27 39 MPO 0 11 1.6 38 40 100 MPO effect of 0.1 N Cl- on DIT-stimulated coupling with MPO. 1 12 1.7 36 40 MPO 100 Table III shows the results of an experiment in which 0 0 5.4 1.1 43 40 TPO the coupling activity of MPO was compared to that of 1 25 38 0 18 4.5 TPO TPO at varying enzyme concentrations. Results for both 1 17 4.2 25 38 TPO 100 bovine and human MPO are presented. Only DIT-stimNote. The labeled Tg was prepared by nonenzymatic iodination of ulated coupling was measured. Coupling increased only slightly with increasing concentration of enzyme, and the goiter Tg with ‘*‘I;, as described in the Materials and Methods section. The initial labeled Tg contained 23.5 atoms I/molecule and the ‘*‘Iactivity of both h-MPO and b-MPO compared favorably labeled iodoamino acid distribution was 1.5% Th, 0.25% Ts, 52% DIT, with that of TPO at all concentrations. and 40% MIT. The initial labeled iodoamino acid distribution was deComparison of effect of Br- and Cl- on MPO-catalyzed iodination. As shown in Fig. 3,0.1 N Br- was even more
effective than 0.1 N Cl- in stimulating MPO-catalyzed iodination. The iodine acceptor in this experiment was BSA, and the HzOz concentration was 300 PM. The pH
termined after pronase digestion of labeled Tg incubated with glucoseglucose oxidase alone. The coupling incubation system contained 1.5 gM labeled Tg, 25 nM MPO or TPO, 0 or 100 mM NaCl, 0 or 1 pM DIT, 1 mg/ml glucose, and 0.1 rg/ml glucose oxidase, in 65 mM phosphate buffer, pH 7.0. The reaction was initiated with glucose oxidase, and samples were incubated for 20 min at 37°C.
MYELOPEROXIDASE-CATALYZED
IODINATION
occurs, we tested iodination of BSA and goiter Tg with NaOCl at pH 5.4 (Fig. 4). At this pH OCll exists almost entirely as HOC1 (p& = 7.5). In an incubation mixture containing 7.5 PM BSA and 100 PM lz51-, addition of 300 FM NaOCl produced binding of 77% of the 1251in 1 min. With 1 PM goiter Tg as the acceptor, 46% of the 1251was similarly bound. Iodination occurred even more readily under these conditions than in the MPO-catalyzed reactions of Fig. 1 and Fig. 3. As in the MPO-catalyzed reaction, BSA was more readily iodinated than goiter Tg. These results provide a possible explanation for the stimulatory effect of Cl- on MPO-catalyzed iodination of protein at pH 5.4, observed in Figs. 1 and 3. Effectof thioureylene drugs on MPO-catalyzed i&nation of BSA. The thioureylene antithyroid drugs, B-propylthiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MMI, methimazole) are well known inhibitors of TPOcatalyzed iodination. Figure 5 shows the effect of varying concentrations of PTU and MM1 on MPO-catalyzed iodination of BSA. The results with MPO show some of the same features that we have previously reported (33, 34) for TPO-catalyzed iodination (see Discussion section). DISCUSSION TPO was the first animal peroxidase whose primary amino acid sequence was deduced from its nucleotide sequence (3-6). Shortly thereafter, the primary amino acid
TABLE Effect
of Enzyme with
III
Concentration on Coupling MPO and TPO Labeled iodoamino acid distribution after coupling % of i3iI in pronase digest
Enzyme h-MPO
b-MPO
TPO
Enzyme concn. (nM)
T4
T3
DIT
MIT
5 12.5 25 5 12.5 25 5 12.5 25
11 13 14 12 13 14 13 15 16
1.5 2.1 2.6 1.8 2.5 3.2 2.1 3.4 3.8
37 33 30 34 31 28 34 28 26
43 43 43 43 42 42 42 41 40
Note. The labeled Tg was prepared by nonenzymatic iodination of goiter Tg with r3*I;, as described in the Methods and Materials section. The I’iI-Tg initially contained 26.3 atoms I/molecule and the following i3iI distribution: Tp, 1.8%; Ts, 0.24%; DIT, 50%; MIT, 42%. The coupling incubation system contained 1.44 j&M labeled Tg, 1 PM DIT, 1 mg/ml glucose, and 0.1 fig/ml glucose oxidase, in 65 mM phosphate buffer, pH 7.0. The reaction was initiated with glucose oxidase, and all samples were incubated for 20 min at 37°C. Each value is the mean of closely agreeing duplicates.
AND
243
COUPLING
so 70 t
u 602 ‘E 50e 0
40-
!
30-
3 20 IOOfI
I
1
I
I
1
I
I
1
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
FIG. 3. MPO-catalyzed iodination of BSA at various pHs; effect of 0.1 N Cl- and 0.1 N Br-. See legend to Fig. 1. The effect of 0.1 N Br- on pH was very similar to that of 0.1 N Cl-.
sequence of MPO was determined in the same manner (7, 8), and more recently that of LPO (9). Before the sequence of LPO was published, Kimura and Ikeda-Saito (35) compared the sequences of TPO and MPO and concluded that, even though the two enzymes have distinctly different physiological functions, they are evolutionarily related members of the same gene family. The more recent data with LPO suggests that it too is a member of the same gene family. Both MPO and LPO show significant cross reactivity with autoantibodies to TPO, which are frequently found in serum from patients with autoimmune thyroid disease (36). It was early demonstrated that purified MPO catalyzes iodination of tyrosine (ll), BSA (12), and thyroglobulin.4 It has also been shown (13) that MPO, covalently bound to Sepharose, can be used to iodinate a variety of proteins. MPO is a major component of the oxygen-dependent bactericidal system in leucocytes, and its ability to catalyze iodination forms the basis of a screening test for cellular or humoral abnormalities in the phagocytic process (14). In the present study we focused primarily on MPOcatalyzed iodination of Tg and on the ability of MPO to catalyze the formation of thyroxine. Aside from a preliminary observation reported many years ago,4 it was not known whether MPO, like TPO and LPO, catalyzes the coupling reaction. Iodination and coupling are very different types of chemical reaction and almost certainly involve different reaction mechanisms. In this connection, ’ Taurog, A., and Gamble, D. E. (1966) Fed. Proc. 26.347.
[Abstract]
244
TAUROG
90
1
SOI
;
50
AND
BSA
/
r-O
goiterTg
40
1oopM ‘2513OOpM NaOCl
30
67 mM phosphatebuffer, pH
5.4
room temperature
Ilr
“y, ( ( , , 1
2
3
4
5
incubation Time (min) FIG. 4. Nonenzymatic iodination of BSA and goiter Tg with NaOCl. The incubation mixture contained either 7.5 pM BSA or 1 pM goiter Tg. Other conditions were as indicated in the figure. The reaction was initiated with NaOCl and stopped after, 1,3, or 5 min by the addition of sodium thiosulfate (final concentration, 10 mM). Protein-bound iodine was separated from unreacted iodide by paper chromatography.
it is of interest that cytochrome c peroxidase readily catalyzes the coupling reaction, but shows no activity in the iodination of Tg or BSA [(37), Taurog and Dorris, unpublished observations]. We were unable to find in the literature a systematic study of the effect of pH on MPO-catalyzed iodination of protein. Also, in preliminary experiments reported many years ago (12), we observed that 0.1 N Cl- had a marked stimulatory effect on MPO-catalyzed iodination of BSA. In our present study, therefore, involving MPO-catalyzed iodination of goiter Tg or BSA, we first examined the effects of pH and 0.1 N Cl-. Iodination was measured 1 min after the reaction was initiated with H202 to approximate initial rates of iodination. Comparable studies were performed with TPO and LPO. MPO-catalyzed iodination of Tg was markedly affected by pH and by 0.1 N Cl- or Br-. In the absence of Cl- or Br- the pH optimum for iodination of Tg was about 4.0, much lower than that observed for LPO (5.4) or for TPO (6.6). In the presence of 0.1 N Cl-, the pH optimum for MPO-catalyzed iodination shifted to about 5.5. At this pH 0.1 N Cl- markedly stimulated the rate of iodination. At pH 4.0, the optimum pH in the absence of Cl-, the addition of 0.1 N Cl- inhibited iodination by more than 98%. This is consistent with the model proposed by An-
DORRIS
drews and Krinsky (26), suggesting two distinct Cl- binding sites on MPO, one acting as a substrate site for oxidation of Cl- to HOC1 and the other as an inhibitory site. According to their model, the latter site requires prior protonation and has an estimated pK, of about 4.5. Our observation that MPO-catalyzed iodination is markedly inhibited by 0.1 N Br- at pH 4.0 (Fig. 3) suggests that the Cl- inhibitory site is also sensitive to excess Br-. We attempted to compare Km values for MPO- and TPO-catalyzed iodination of Tg and BSA by measuring initial rates of iodination at varying concentrations of iodide. However, in the case of MPO we observed a lag phase in the kinetics of iodination, in both the presence and absence of Cl-. Such a lag was not observed with TPO or LPO. We were unable, therefore, to obtain reliable measurements of initial rates of MPO-catalyzed iodination. At present, we have no adequate explanation for this lag phase and until this problem is resolved, we are unable to determine a meaningful K,,, value for MPOcatalyzed iodination. The marked stimulatory and inhibitory effects of Clon MPO-catalyzed iodination were not observed with TPO or with LPO. A major difference between MPO and TPO or LPO is that MPO is capable of oxidizing Cl- to HOCl. The pH optimum of this reaction is about 5.5 (31), very close to the pH optimum of Cl--stimulated, MPOcatalyzed iodination of goiter Tg and BSA observed in the present study (Figs. 1 and 3). These results suggested that the stimulatory effect of Cl- involves oxidation of Iby HOC1 to an iodinating species (e.g., HOI) that can iodinate tyrosyl residues of protein nonenzymatically. Evidence for this was obtained by our demonstration (Fig. 4) that BSA and goiter Tg are readily iodinated by a mixture of NaOCl and I- at pH 5.4 in the absence of peroxidase or HzOz. At this pH OCll exists primarily as HOCl.
Glucose 1 mgml Glumse Oxidase 0.5 tin-d
T) s 8
80
8o
%
40
10
20
30
40
80
80 Incubation
Time (min)
FIG. 5. Effect of antithyroid drugs, MM1 and PTU, on MPO-catalyzed iodination of BSA. b-MPO was used in this experiment. Similar results were obtained with h-MPO.
MYELOPEROXIDASE-CATALYZED
We have previously suggested (38) that the function of TPO and LPO in iodination of tyrosine and tyrosyl residues in protein may be to generate HOI, which could then iodinate nonenzymatically. A similar proposal was made for HRP-catalyzed iodination of tyrosine (39). In a study performed many years ago (40) with chloroperoxidase (CPO), we observed that CPO-catalyzed iodination of tyrosine and thyroglobulin was even more markedly stimulated by 0.1 N Cl- than was MPO-catalyzed iodination in the present study. We suggested that an oxidized form of Cl- was an intermediate in oxidizing I- to an iodinating species. It now appears likely that HOC1 was the intermediate involved in our previously observed stimulatory effect of Cl- on CPO-catalyzed iodination. For studying T4 formation in the MPO-catalyzed iodination of Tg, we used an incubation system at pH 7.0 containing the HzOz generating system, glucose-glucose oxidase (Fig. 2), and an incubation period of 45 min. The iodinating activity under these conditions was also stimulated by 0.1 N Cl-. The mechanism of this stimulatory effect may also be mediated through the formation of HOCl/OCll. We have shown previously (38) that the steady state level of HzOz during TPO-catalyzed iodination in the presence of glucose-glucose oxidase is very low, especially when compared with the 100 PM H202 added for the 1-min incubations in Fig. 1. Thus, the Cl-/ H202 ratio was much greater in the system containing glucose-glucose oxidase (Fig. 2) than it was under the conditions used in Fig. 1. As shown by previous investigators (31, 41), this would shift the pH optimum of Cloxidation to more alkaline pHs. It is likely, therefore, that under the conditions used in Fig. 2 (pH 7.0, glucose-glucose oxidase), there was significant oxidation of Cl- to HOCl/OCll by MPO. There was also very significant formation of T*, comparable to that observed in a similar incubation system containing TPO, with or without Cl-. MPO was also tested in a coupling system designed to measure T4 formation in the absence of iodination (Tables 2 and 3). In the absence of free DIT it was more active than TPO on a molar basis under the conditions used in Table II. However, in the presence of a low concentration of free DIT, which acts to stimulate the coupling reaction, TPO was more active than MPO. We have previously proposed a radical mechanism for the TPO-catalyzed coupling reaction (l), based on suggestions of previous investigators (43,44). The same mechanism may also apply to MPO-catalyzed coupling. Peroxidase-catalyzed iodination, on the other hand, is generally regarded as a two-electron reaction (l), in which I- is oxidized directly by Compound I to I+ or its equivalent oxidation state, HOI. MPO-catalyzed iodination was inhibited by the thioureylene antithyroid drugs, MM1 and PTU, in a manner similar to that previously observed for TPO-catalyzed iodination (33, 34). At lower drug concentrations (Fig. 5),
IODINATION
AND
245
COUPLING
inhibition of iodination was transient, and after a variable interval, dependent on the drug concentration, there was escape from inhibition. In the case of TPO, we have shown that the period of inhibition is associated with extensive, iodide-dependent, drug oxidation. At higher drug concentrations, however, the enzyme is rapidly inactivated by the drug, and only limited drug oxidation occurs before the enzyme is inactivated. We have previously (34) proposed a scheme to explain reversible and irreversible inhibition of TPO-catalyzed iodination by MM1 and PTU. Although only limited data were obtained for inhibition of MPO-catalyzed iodination by MM1 and PTU, the results in Fig. 5 suggest that the same general scheme may also apply to MPO-catalyzed iodination. REFERENCES 1. Taurog, A. (1991) in Werner’s The Thyroid (Braverman, L. E., and Utiger, R. D., Eds.), 6th ed., pp. 51-97, J. B. Lippincott, Philadelphia. 2. Taurog, A., Dorris, M. L., and Lamas, L. (1974) Endocrinology 1286-1294.
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