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TRH-TSH-THYROID |
Department of Internal Medicine, Erasmus University Medical Center, 3000 DR Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Theo J. Visser, Ph.D., Department of Internal Medicine, Room Bd 234, Erasmus Medical Center, Dr. Molewaterplein 40, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: . visser{at}inw3.azr.nl
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COS cells were transfected with expression vectors encoding wild-type (wt) D2, D2 A131C, or D2 A131S proteins. Kinetic analysis was performed on homogenates with dithiothreitol (DTT) as reducing cofactor. The D2 A131C and A131S mutants displayed similar Michaelis-Menten constant values for T4 (5 nM) and reverse T3 (9 nM) as the wt D2 enzyme. The limiting Michaelis-Menten constant for DTT of the D2 A131C enzyme was 3-fold lower than that of the wt D2 enzyme. The wt and mutant D2 enzymes are essentially insensitive to propylthiouracil [concentration inhibiting 50% of activity (IC50) > 2 mM] in the presence of 20 mM DTT, but when tested in the presence of 0.2 mM DTT the IC50 value for propylthiouracil is reduced to about 0.1 mM. During incubations of intact COS cells expressing wt D2, D2 A131C, or D2 A131S, addition of increasing amounts of unlabeled T4 resulted in the saturation of [125I]T4 deiodination, as reflected in a decrease of [125I]T3 release into the medium. Saturation first appeared at medium T4 concentrations between 1 and 10 nM.
In conclusion: substitution of cysteine for a conserved alanine residue in the catalytic center of the D2 protein does not inactivate the enzyme in vitro and in situ, but rather improves the interaction with the reducing cofactor DTT in vitro.
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5 µM), is activated by dithiothreitol (DTT) in vitro, and is highly sensitive to inhibition by propylthiouracil (PTU) in vitro and in vivo. The D2 protein has a low Km for T4 (5 nM), is also activated by DTT but is only minimally sensitive to PTU (8, 9, 10). The catalytic mechanism, though incompletely described, seems to be different between D1 and D2. ORD of T4 by D1 protein exhibits ping-pong (bi)-substrate kinetics with T4 and the thiol-containing cofactor as cosubstrates (6, 8, 11, 12). The D2 protein exhibits sequential reaction kinetics, suggesting that T4 and the thiol-containing cofactor must interact with the enzyme simultaneously before reaction takes place (8, 9). Another remarkable difference between D1 and D2 is the fact that while N-bromoacetyl derivatives of T4 or T3 are excellent covalent affinity labels of D1, they do not label the D2 selenoprotein (13, 14, 15, 16). The D1 protein is a selenoenzyme, containing a selenocysteine (SeC) residue in the catalytic center (4, 17, 18). This SeC residue is essential for enzymatic activity because replacement with leucine or alanine eliminates deiodinase activity, whereas replacement with cysteine reduces the turnover number about 100-fold (19, 20). The SeC in the catalytic center of D1 is thought to function as the iodine acceptor and PTU probably inhibits D1 via the formation of a SeC-PTU adduct (6, 12, 19). The inability to covalently label D2 with bromoacetyl-iodothyronine derivatives, as well as the drastically reduced sensitivity to PTU, iodoacetate, and gold thioglucose compared with D1, have been interpreted as evidence that deiodination by D2 does not require the participation of enzyme sulfydryl groups (21) or that D2 is not a selenoprotein (19, 22). The inability to label the substrate-binding subunit of D2 (p29) in rat glial cells with 75Se was also interpreted as evidence that D2 is not a selenoprotein (23). However, since the cloning of D2 cDNAs from several species and the identification of SeC insertion sequence elements (SECIS) in the 3'-UTR, it is clear that the D2 protein is a selenoenzyme containing a SeC residue in the catalytic center (10, 16, 24, 25, 26, 27, 28, 29). More direct evidence for the selenoprotein character of D2 protein was recently provided by the demonstration that a 75Se-labeled protein of the expected size is immunoprecipitated from mesothelioma cells with a D2 antiserum (30). Therefore, the described differences between D1 and D2 must be influenced by other factor(s) than the presence of SeC in the catalytic center.
The core catalytic center of D1 and D2 is located in a region about 15 amino acids long, surrounding the essential SeC. This region is highly conserved within and between the D1 and D2 subtype enzymes (Fig. 1
). However, all of the D2 proteins cloned so far have an alanine two residues amino-terminal to the SeC, whereas all D1 proteins contain a cysteine (Cys) at this position (1, 2, 3, 29). A Cys residue in the catalytic center could assist in enzymatic action by providing a nucleophilic sulfide or by participating in redox reactions with a cofactor or enzyme residues. Replacement of Cys by alanine (Ala) in the active center of D1 caused a marked increase in the limiting Km for DTT (11, 31). By analogy, the absence of a Cys residue at the same position (residue 131) in human D2 might reduce the turnover number compared with D1 and could perhaps explain the minimal sensitivity of D2 to PTU or the inability to react with bromoacetyl derivatives of T3 and T4.
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PTU (6-n-propyl-2-thiouracil), gold thioglucose (GTG), and iodoacetate (IAc) were obtained from Sigma (St. Louis, MO). DTT was obtained from ICN Biochemicals, Inc. (Costa Mesa, CA). Recombinant Taq DNA-polymerase was obtained from Amersham Pharmacia Biotech (Roosendaal, The Netherlands) and synthetic oligonucleotides were ordered from Life Technologies, Inc. (Glasgow, UK).
Site-directed mutagenesis and construction of expression vectors
A cDNA clone containing the entire coding sequence of the human D2 protein was used for mutagenesis via the overlap extension DNA-polymerase reaction procedure (34).
Overlapping inner sense and antisense primers containing the nucleotide changes needed to produce the SeC133Cys (SeC133C) D2 mutant cDNA, Ala131Cys (A131C) D2 mutant cDNA, Ala131Ser (A131S) D2 mutant cDNA, and SeC133Cys/Ala131Cys (double Cys) D2 mutant cDNA were used for amplification with a Kozak start consensus (underlined) 5'-outer primer (5'-CAAGCTTGCCACCATGGGCATCCTCAGCGTA) or a stop 3'-outer primer (5'-CAAGCTTTTAACCAGCTAATCTAGT), each containing flanking HindIII restriction sites (italics). The PCR products were agarose gel purified and used for overlap extension PCR reactions with the described outer primers. The mutated D2 cDNA fragments were subcloned into the pGEM-T vector (Promega Corp., Madison, WI) and sequenced to confirm that only the proper mutations were introduced. Because a SECIS element is required for incorporation of SeC in selenoproteins, we prepared chimeric constructs in which the D2 cDNA was inserted 5' to the SECIS element of the rat D1 gene. For this purpose, the G21-pcDNA3 rat D1 expression vector (4) was digested with HindIII, and the 6-kb DNA band containing vector DNA plus 0.7 kb of the rat D1 3'-UTR (including the SECIS element) was isolated from a preparative agarose gel. The pGEM vectors containing the respective mutant D2 sequences were digested with HindIII, and the isolated fragments were cloned into the prepared rat D1-SECIS-pcDNA3 vector. A wt human D2 expression vector was prepared in the same manner. It should be noted that the 3'-(second) TGA, which is seven codons 5' to the universal stop codon TAA in the D2 cDNA, is present in all constructs. It is not known how efficient the second UGA codon is translated as SeC.
Expression of D2 protein
The wt D2 and mutant D2 enzymes were expressed in COS-1 cells after diethylaminoethyl-dextran-mediated transfection of the expression vectors (35). COS-1 cells were grown in DMEM-Ham’s F-12 medium (DMEM/F12) containing 10% FCS (Life Technologies, Inc.) and 40 nM Na2SeO3. One day before transfection, cells were divided 1:10 in 90-mm diameter culture dishes. Expression constructs (8 µg per dish), isolated by alkaline lysis and polyethyleneglycol precipitation (36), were added to 5 ml serum-free DMEM/F12 medium containing 200 µg/ml diethylaminoethyl-dextran. After 2 h, the medium was replaced by serum-free DMEM/F12 containing 100 µM chloroquine phosphate (Sigma). Again, 2 h later the medium was replaced with DMEM/F12 containing 10% FCS and 40 nM Na2SeO3. After 2 d, the cells were rinsed with PBS and collected in 0.25 ml 0.1 M phosphate (pH 7.2), 2 mM EDTA buffer, sonicated, aliquoted, and stored at -75 C.
Assay of ORD activity in COS cell homogenates
The principle of this assay is the production of radioiodide by ORD of [3', 5'-125I]T4 or [3', 5'-125I]rT3. Duplicate incubations contained about 100,000 cpm labeled T4 or rT3 with varying amounts of unlabeled substrate (T4 or rT3) and COS cell homogenates (50–100 µg protein) in a final volume of 0.2 ml P100E2D20 buffer (0.1 M sodium phosphate; 2 mM EDTA; 20 mM DTT, pH 7.2). In some experiments, the DTT concentration was varied between 0.1 and 20 mM DTT. In various experiments, the inhibitors PTU (0.1–2 mM), GTG (0.1–3 µM), and BrAcT4 (0.1–1000 nM) were included. Mixtures were incubated for 60 min at 37 C, whereafter the reaction was stopped by the addition of 0.1 ml 5% BSA on ice. Protein-bound iodothyronines were subsequently precipitated by 10% TCA on ice, and the radioiodide in the supernatant was separated from remaining iodothyronines by chromatography on Sephadex LH-20 mini columns as described (33). The fractions were counted for 125I-activity in a 16 channel 
-counter (NE1600, Nuclear Enterprises, Edinburgh, UK).
Enzymatic deiodination was corrected for nonenzymatic deiodination in blank incubations with homogenates of nontransfected COS cells (< 3% of total deiodination). Protein was adjusted to consume less than 30% of substrate, and in control experiments it was determined that the deiodination rate was linear up to 60 min incubation. The radioiodide production counted was multiplied by 2 to account for the random labeling and deiodination at the 3' and 5' positions in the substrate. By reverse-phase HPLC of reaction products, it was established that equal amounts of radioiodide and radioactive iodothyronine (T3 or T2) were produced. No inner-ring deiodination was detected with wt or D2 mutant enzymes.
Assay of ORD activity in intact transfected COS cells
COS cells were cultured in 6-well plates and transfected with 2.5 µg plasmid per well as described. One day after transfection, cell monolayers were washed with serum-free DMEM/F12 medium and then cultured for an additional 24 h in serum-free DMEM/F12 (plus 40 nM Na2SeO3), to which was added [125I]T4 (1 x 106 cpm/ml) plus 1–1000 nM unlabeled T4.
Medium was harvested and extracted with ice-cold methanol (1:1). After centrifugation, the extract was mixed with 0.02 M ammonium acetate (pH 4) and 0.1 ml of the mixture was applied to a 250 x 4.6 mm Symmetry C18 column connected to an Alliance HPLC system (Waters Chromatography Division, Millipore Corp., Milford, MA), and eluted with a 20-min gradient of 24–29% acetonitrile followed by a 6-min gradient of 29–50% acetonitrile in 0.02 M ammonium acetate (pH 4) at a flow rate of 1.2 ml/min. Radioactivity in the eluate was monitored on line using a Radiomatic A-500 flow scintillation detector (Packard, Meriden, CT). The percentage T4 deiodination was calculated from the amount of [125I]T3 produced (multiplied by two), divided by the amount of [125I]T4 added.
Incubation with N-bromoacetyl-[125I]T3 or T4
Labeling of COS cell homogenates was done according to previously published procedures (14). In short, solutions of BrAc[125I]T3 or BrAc[125I]T4 (150,000 cpm) in ethanol were pipetted into microcentrifuge tubes, and the solvent was evaporated by a stream of nitrogen. After addition of 25 µl P100E2D3 [100 mM sodium phosphate; 2 mM EDTA; 3 mM DTT, pH 7.2], and vortexing, the COS cell homogenates (150 µg protein) were added in a total volume of 50 µl P100E2D3. Again the mixtures were vortexed and incubated at 37 C for 20 min. Reactions were terminated by the addition of gel-loading buffer, and samples were analyzed by SDS-PAGE (12% gel) followed by autoradiography (14).
For in situ affinity labeling experiments, COS cells were transfected with wt D2, D2 A131C, or rat D1 expression vectors, and 1 d after transfection cells were washed and than cultured for an additional 24 h in serum-free DMEM/F12, to which was added BrAc[125I]T4 (200,000 cpm/ml equivalent to 0.25 nM). The medium was harvested and analyzed by HPLC as described. The cells were collected in 0.25 ml 0.1 M phosphate (pH 7.2), 2 mM EDTA buffer, sonicated, and samples were analyzed by SDS-PAGE.
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COS cells were transfected with the described expression vectors and kinetic analysis was performed on homogenates in the presence of 20 mM DTT (Fig. 2 and Table 1). The wild-type D2, D2 A131C, and D2 A131S enzymes displayed similar Km values for T4 (5 nM) as well as for rT3 (9 nM). Remarkably, substituting Cys for SeCys caused a 1,000-fold higher Km for the substrate T4 and a 10-fold higher Vmax compared with wt D2 protein. The same holds true for the double Cys mutant (D2 SeC133C/A131C) enzyme. Coincubations with up to 250 µM selenocystine (which is converted to selenocysteine in the presence of DTT) did not further increase the deiodination rate of the D2 SeC133C enzyme (not shown).
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The D2 protein exhibits sequential reaction kinetics, and Lineweaver-Burk plots at different reducing cofactor (DTT) concentrations yield a set of intersecting lines (8, 9). To investigate the possibility that introduction of a Cys in the catalytic center influences the interaction with reducing cofactor, T4 ORD was studied at several concentrations of DTT. The transfected COS cells were harvested in phosphate buffer (pH 7.2) without DTT, and without addition of DTT the net deiodination of T4 was less than 0.5% in 60 min. In initial experiments, the T4 concentration was fixed (2 nM) and the DTT concentration was varied over a broad range (0.1–30 mM). At the lowest DTT concentrations (0.1 and 0.3 mM), the deiodination rate of the D2 A131C enzyme was higher compared with the D2 A131S and D2 wt enzymes (Fig. 3). In more detailed studies, both DTT (0.1–1 mM) and T4 (1–10 nM) concentrations were varied. The Lineweaver-Burk plots were derived at each DTT concentration for the wt D2 and D2 A131C enzyme (Fig. 4, A and B, respectively). The plots consisted of a set of intersecting lines, implying that the sequential mechanism of deiodination is not altered by the introduction of a Cys residue in the catalytic center. A replot of the reciprocal of the apparent Vmax values obtained against the reciprocal of the DTT concentrations yielded the limiting Km for DTT. The limiting Km DTT for the D2 A131C enzyme was 3-fold lower than that of the wt D2 enzyme (0.10 mM ± 0.03 vs. 0.37 mM ± 0.15, respectively, mean ± SEM, n = 3). The lower limiting Km DTT of the D2 A131C enzyme would predict a higher Vmax compared with wt D2 enzyme at a DTT concentration of 0.1 mM, provided that the expression levels are equal (Fig. 5). Indeed, under these conditions the Vmax of the D2 A131C enzyme was higher compared with the wt D2 enzyme (0.10 pmol/min·mg vs. 0.06 pmol/min·mg), whereas the Km for T4 was similar (0.7 nM).
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DTT is a potent but nonphysiological reducing cofactor for D2 (8, 9). The monothiol glutathione (GSH) might be a physiological cofactor for D2. However, neither the wt D2, the D2 A131C, nor the D2 A131S enzymes are activated by GSH (range 0.1–25 mM tested) in vitro (not shown).
To compare the PTU sensitivity of the wt D2, D2 A131C, and D2 A131S enzymes, the deiodination of T4 was studied in the presence of different concentrations of the reducing cofactor DTT because it has been shown that PTU inhibits D2 activity in brown adipose tissue homogenates at low DTT (<5 mM DTT) concentrations (9). In the presence of 20 mM DTT, no significant inhibition by PTU was observed (Fig. 6A). However, in the presence of 0.2 mM DTT the enzyme activity was about 70% inhibited by 1 mM PTU for the wt D2, D2 A131C, and D2 A131S enzymes (Fig. 6A). In the presence of 0.2 mM DTT, the wt D2 and D2 A131S enzymes are about 40% as active as in the presence of 20 mM DTT, whereas the D2 A131C enzyme is about 70% as active as in the presence of 20 mM DTT. We have not investigated the inhibitory mechanism but it is unlikely that the inhibition is caused by a reaction of (oxidized) PTU with DTT because even at a PTU concentration of 2 mM, the inhibition was not complete.
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) of the D2 A131C and D2 A131S enzymes in comparison with wt D2 could be detected (IC50 0.7 µM). IAc is a weak inhibitor of native D2 (IC50 = 1000 µM) but a strong inhibitor of native D1 (IC50 = 2 µM), which has a Cys in the catalytic center (21, 39). However, the sensitivity of D2 for IAc was not increased by the introduction of a Cys residue in the catalytic center. For both the wt D2 and D2 A131C enzymes, the IC50 was greater than 1000 µM (not shown).
Wild-type D2, D2 A131C, and D2 A131S proteins are not covalently labeled by N-bromoacetyl-[125I]T3 or N-bromoacetyl-[125I]T4
When homogenates from COS cells expressing wt D2, D2 A131C, or D2 A131S protein were incubated with BrAc[125I]T3 or BrAc[125I]T4, the labeling patterns obtained after SDS-PAGE were identical with those obtained using homogenates of nontransfected COS cells (not shown). Under identical experimental conditions, labeling patterns of homogenates from COS cells expressing rat D1 protein showed an additional labeled band of 29 kDa corresponding to the D1 protein (not shown). Analysis by SDS-PAGE of labeling patterns from in situ labeled COS cells revealed labeling of the rat D1 protein (>5 d autoradiography), but no labeling of wt D2 or D2 A131C protein. More than 90% of the added radioactivity was recovered in the medium, and by HPLC analysis it was found that almost all BrAc[125I]T3 was converted to more polar compound(s). This conversion was not further investigated, but it may reflect reaction with free amino acids or GSH.
Despite the fact that BrAcT4 does not bind covalently to D2 protein, it does inhibit T4 deiodination of wt D2, D2 A131C, and D2 A131S enzymes (Fig. 6C). The inhibition is complete at 100 nM BrAcT4, and the IC50 is about 0.5 nM.
Deiodination by wild-type D2, Ala131Cys, and Ala131Ser D2 mutants in intact cells
The D2 A131C and D2 A131S enzymes are active in vitro in the presence of the cofactor DTT. An important question is if the same is true under in situ conditions using intact transfected cells, in which the availability of T4 and/or cofactor activity may be more restricted.
When wt D2 was expressed in COS cells, addition of increasing amounts of unlabeled T4 resulted in the saturation of [125I]T4 deiodination, as reflected in a decrease of [125I]T3 release into the medium (Fig. 7). Saturation first appeared at medium total T4 concentrations between 1 and 10 nM (the radiolabeled T4 contributes about 0.5 nM). In control experiments using [125I]T4, it was established that nontransfected COS cells contain no deiodinase activity nor sulfotransferase activity. When the D2 A131C and D2 A131S enzymes were studied in the same manner, there was a similar progressive decrease in the fractional deiodination of [125I]T4, thus indicating that the D2 mutants are active under in situ conditions (Fig. 7).
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Indeed, the D2 A131C protein is enzymatically active. The most striking finding was the reduced limiting Km for the cosubstrate DTT of the D2 A131C enzyme, whereas the Km values for the substrates T4 and rT3 were similar to those of wt D2. The rather low Km for DTT of the wt D2, D2 A131C, and D2 A131S enzymes are perhaps surprising given the fact that typical D2 assays are usually done in the presence of 20 mM DTT. In many instances, D2 assays are done on microsomal fractions, and under those conditions the limiting Km for DTT is greater than 10 mM (1, 8). Using brown adipose tissue homogenates, Goswami et al. (9) reported a limiting Km DTT of 0.5 mM for D2, which is in line with our results using COS cell homogenates. The Km values for T4 and rT3 (5 nM and 9 nM, respectively) determined in the presence of 20 mM DTT are in agreement with previously reported values using homogenates of transfected cells (10, 16, 25). In the presence of 0.1 mM DTT, the Km for T4 is reduced to 0.7 nM, which is inherent to the sequential reaction kinetics of the D2 enzyme.
The Vmax of D2 A131C enzyme is about 2-fold lower than that of wt D2 and D2 A131S enzymes, when tested in the presence of 20 mM DTT (Table 1). We performed semiquantitative Western blotting of COS cell homogenates with polyclonal antisera directed against the C terminus or the catalytic center of rat D2 protein (37). Unfortunately, only in homogenates containing D2 SeC133C or D2 SeC133C/A131C protein, a specific band with the expected molecular mass (31 kDa) could be detected (not shown). It was shown before, using different antisera, that the expression level of the human D2 SeC133C protein is about 100-fold higher than that of wt D2 protein in transfected cells (38). The wt D2 and D2 A131C protein levels in COS cell homogenates are too low and/or the cross-reactivity of the rat D2 antisera with human D2 protein is not strong enough. Consequently, we cannot answer the question whether the 2-fold lower Vmax of the D2 A131C enzyme is due to reduced protein levels or reduced catalytic efficiency in the presence of 20 mM DTT. Remarkably, when tested in the presence of 0.1 mM DTT, the Vmax of D2 A131C enzyme is 2-fold higher compared with wt D2. It has been shown that modest changes in deiodination kinetics resulting from mutations in the catalytic center of D1 enzyme were not apparent when the same mutant constructs were tested in intact cell systems (31). On the other hand, the large (1,000-fold) increase in the Km for T4 due to the replacement of SeC133 by Cys in D2 was reflected in assays with intact transfected cells (38). In our experiments, the modest increase in Vmax of the D2 A131C enzyme compared with the wt D2 enzyme in the presence of 0.1 mM DTT was not reflected in the intact cell system. Limitations in the rate of cellular T4 uptake or endogenous cofactor availability could restrict the rate of deiodination and thus negate a difference in the activities of the D2 enzymes. It is clear, however, that introduction of a Cys residue in the catalytic center of D2 protein does not impair catalytic activity in vitro and in situ.
The D1 protein is strongly inhibited by PTU (IC50 = 5 µM), GTG (IC50 = 0.05 µM), and IAc (IC50 = 2 µM), and this was initially explained by the presence of a reactive SH group in the enzyme-active center (6, 7, 8). When D1 protein was identified as a selenoprotein, it was proposed that the SeC residue reacts in the reduced (SeH) form with IAc and GTG, whereas the selenenyl iodide (SeI) intermediate is the target for inhibition by PTU (19, 39). The D2 enzyme also contains a SeC residue in the catalytic center but is much less sensitive to inhibition by PTU (IC50 > 1000 µM), GTG (IC50 = 1 µM) and IAc (IC50 = 1,000 µM) when tested in the presence of 20 mM DTT. Only when tested in the presence of 0.2 mM DTT, the D2 enzyme becomes more sensitive to PTU. Introduction of a cysteine residue in the catalytic center (D2 A131C), as is the case in D1, did not significantly increase the sensitivity of D2 for PTU, GTG, or IAc. This suggests that the wt D2 and D2 A131C enzymes use a different mechanism of deiodination than D1. This conclusion is supported further by the fact that both the wt and D2 A131C enzymes appear to follow sequential reaction kinetics, whereas D1 follows ping-pong kinetics (8, 11, 19, 39).
Remarkably, substituting Cys for SeCys133 caused a 1,000-fold higher Km for the substrate T4 (Table 1), in line with previously published data (38). The same holds true for the double Cys mutant (D2 SeC133C/A131C) protein. In contrast, substitution of Cys for SeCys in D1 caused a modest 5- to 10-fold increase in the Km for ORD of rT3 and T4 (20).
We propose a model in which the SeC residue in D2 exerts a nucleophilic attack to the 2'-position of T4, thus forming a bond between the SeC residue and T4 (Fig. 8). Subsequent steps involve the abstraction of iodonium (I+) by DTT (or another thiol group containing cofactor), providing a D2·T3 complex and a cofactor sulfenyl iodide. The latter will rapidly disproportionate to iodide (I-) and oxidized thiol, whereas the enzyme intermediate will yield T3 and the unmodified enzyme. The reaction as a whole requires two thiol groups, which could be supplied by one DTT molecule. In this model, the activated substrate is directly reduced by the cofactor, whereas in the case of D1 the SeI intermediate is reduced by cofactor (6, 8, 11, 21). In the case of the D2 A131C enzyme, an additional pathway is possible in which the iodonium is transferred to the SH group of Cys 131, followed by reduction of the SI intermediate by DTT.
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In D1, the SeI intermediate is the target for inhibition by PTU (6, 12, 19, 39, 43). According to our model, such an intermediate does not exist in D2, thus explaining the insensitivity for PTU of D2. Only under suboptimal conditions causing reduced deiodination rates, i.e. at low (0.2 mM DTT) reducing cofactor concentrations, the wt D2 and D2 mutant enzymes become sensitive to PTU, although the inhibition is never complete. Under these suboptimal conditions (oxidized) PTU might compete with substrate for reaction with the selenol group. Obviously, this is less favored than reaction with the selenenyl iodide intermediate in D1 explaining the much higher concentrations of PTU needed. The proposed model also explains why the D2 A131C mutant enzyme is not more sensitive to PTU because the D2 A131C enzyme has in fact a higher deiodination rate at low DTT concentrations.
The PTU sensitivity of D2 enzyme at low (and perhaps physiologically more relevant) thiol cofactor concentrations as demonstrated in this and previous studies (9, 46), poses questions with regard to the extent to which D1 and D2 are inhibited in vivo by PTU. Certainly because D1 inhibition by PTU is dependent on turnover (formation of SeI intermediate), which might be limited at physiological T4 concentrations. On the other hand, PTU is taken up in organs that express D1 (liver, kidney), whereas it is not or poorly taken up in several D2-expressing tissues such as brain and brown adipose tissue (46). In line with this, kinetic analysis of circulating T3 in PTU-blocked rats has suggested significant T3 production from T4 by D2 enzyme (47).
The proposed catalytic model might form the basis for the design of transition state analogs that specifically inhibit D2. For instance, brominated analogs of T4 may still bind to D2 and react with the selenol group but may be blocked in bromonium abstraction. Such analogs would permanently block the catalytic site and could in fact also be used as D2 affinity labels.
A bit puzzling still is the low susceptibility of D2 to IAc and GTG. It has been suggested that the reactivity of the selenol group in D2 is much less than that in D1 (39), implying a lower turnover number of D2 enzyme. At the moment, it is not possible to investigate this hypothesis because we have no way to determine the D2 enzyme turnover number. The inhibition by GTG of D2 is noncompetitive with substrate, whereas it is competitive with substrate for D1 (22, 38). The interaction with GTG might therefore occur with a Cys residue outside the catalytic center of D2. An alternative explanation may be that in D1 the modification of the selenol group by GTG or IAc is irreversible, whereas in D2 it is reversible, at least for IAc, because T4 also forms a covalent complex with the selenol group.
A remarkable difference between D1 and D2 protein is the fact that while N-bromoacetyl derivatives of T4 and T3 are excellent affinity labels of D1, they do not label D2 protein (13, 14, 15, 16). In principle, not only SeC but also cysteine, tyrosine, histidine, lysine, and arginine residues may be modified by bromoacetyl reagents (48). After introduction of a Cys residue in the catalytic center of D2, as in D2 A131C, it is still not possible to obtain affinity labeling with BrAcT4 or BrAcT3. Attempts to develop an in situ affinity labeling system for D2 protein with BrAc-T3 were also unsuccessful. These experiments were based on observations that some proteins, as for instance steroid receptors, are labeled more efficiently with affinity ligands in situ than in homogenates (49). Meanwhile, BrAcT4 does covalently label the 29 kDa substrate-binding subunit, p29, of D2 in rat brain cells (50). The p29 protein is not a selenoprotein (51), and its relationship to the 31-kDa D2 selenodeiodinase used in this study is uncertain. The observed strong inhibition by BrAcT4 of wt D2 and mutant D2 enzymes is in line with previous observations that BrAcT3 inhibits D2 activity (52) and that alanine side chain modified analogs of T4 inhibit D2 activity in glial cell sonicates (53).
It has been known for long that D2 activity is rapidly down-regulated by T4 via a posttranscriptional mechanism that increases the rate of degradation or inactivation of the enzyme (53, 54, 55, 56, 57, 58). It was recently proposed that this substrate-induced loss of D2 activity is due to proteasomal degradation of the enzyme and requires substrate interaction with the catalytic center (58). In other words, the catalytic center must be modified in some way by substrate in order for the ubiquitination machinery to be able to recognize D2 protein. The intermediate complexes formed between the SeC residue and the substrates T4 or rT3 proposed in our catalytic model, may fulfill that role.
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Abbreviations: Ala, Alanine; A131C, D2 mutants with a cysteine two-residue amino terminal to the SeC; A131S, D2 mutants with a serine two-residue amino terminal to the SeC; Cys, cysteine; D1, D2, and D3, human types I, II, and III iodothyronine deiodinase; DMEM/F12, DMEM-Ham’s F-12 medium; DTT, dithiothreitol; GSH, monothiol glutathione; GTG, gold thioglucose; IAc, iodoacetate; Km, Michaelis-Menten constant; ORD, outer ring deiodination; PTU, propylthiouracil; rT3, reverse T3; SeC, selenocysteine; SECIS, SeC insertion sequence elements; SeI, selenenyl iodide; wt, wild-type.
Received September 24, 2001.
Accepted for publication December 14, 2001.
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  K. A. Heemstra, M. R. Soeters, E. Fliers, M. J. Serlie, J. Burggraaf, M. B. van Doorn, A. A. van der Klaauw, J. A. Romijn, J. W. Smit, E. P. Corssmit, et al. Type 2 Iodothyronine Deiodinase in Skeletal Muscle: Effects of Hypothyroidism and Fasting J. Clin. Endocrinol. Metab., June 1, 2009; 94(6): 2144 - 2150. [Abstract] [Full Text] [PDF]
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 E. Silvestri, A. Lombardi, P. de Lange, L. Schiavo, A. Lanni, F. Goglia, T. J. Visser, and M. Moreno Age-related changes in renal and hepatic cellular mechanisms associated with variations in rat serum thyroid hormone levels Am J Physiol Endocrinol Metab, June 1, 2008; 294(6): E1160 - E1168. [Abstract] [Full Text] [PDF]
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 M. S Wagner, S. M Wajner, J. M Dora, and A. L. Maia Regulation of Dio2 gene expression by thyroid hormones in normal and type 1 deiodinase-deficient C3H mice J. Endocrinol., June 1, 2007; 193(3): 435 - 444. [Abstract] [Full Text] [PDF]
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 E. C. H. Friesema, G. G. J. M. Kuiper, J. Jansen, T. J. Visser, and M. H. A. Kester Thyroid Hormone Transport by the Human Monocarboxylate Transporter 8 and Its Rate-Limiting Role in Intracellular Metabolism Mol. Endocrinol., November 1, 2006; 20(11): 2761 - 2772. [Abstract] [Full Text] [PDF]
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 F. W. J. S. Wassen, W. Klootwijk, E. Kaptein, D. J. Duncker, T. J. Visser, and G. G. J. M. Kuiper Characteristics and Thyroid State-Dependent Regulation of Iodothyronine Deiodinases in Pigs Endocrinology, September 1, 2004; 145(9): 4251 - 4263. [Abstract] [Full Text] [PDF]
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C. A. Shepherdley, W. Klootwijk, K. W. Makabe, T. J. Visser, and G. G. J. M. Kuiper An Ascidian Homolog of Vertebrate Iodothyronine Deiodinases Endocrinology, March 1, 2004; 145(3): 1255 - 1268. [Abstract] [Full Text] [PDF]
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G. G. J. M. Kuiper, F. Wassen, W. Klootwijk, H. van Toor, E. Kaptein, and T. J. Visser Molecular Basis for the Substrate Selectivity of Cat Type I Iodothyronine Deiodinase Endocrinology, December 1, 2003; 144(12): 5411 - 5421. [Abstract] [Full Text] [PDF]
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 I. Callebaut, C. Curcio-Morelli, J.-P. Mornon, B. Gereben, C. Buettner, S. Huang, B. Castro, T. L. Fonseca, J. W. Harney, P. R. Larsen, et al. The Iodothyronine Selenodeiodinases Are Thioredoxin-fold Family Proteins Containing a Glycoside Hydrolase Clan GH-A-like Structure J. Biol. Chem., September 19, 2003; 278(38): 36887 - 36896. [Abstract] [Full Text] [PDF]
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R. P. Peeters, H. van Toor, W. Klootwijk, Y. B. de Rijke, G. G. J. M. Kuiper, A. G. Uitterlinden, and T. J. Visser Polymorphisms in Thyroid Hormone Pathway Genes Are Associated with Plasma TSH and Iodothyronine Levels in Healthy Subjects J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2880 - 2888. [Abstract] [Full Text] [PDF]
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G. G. J. M. Kuiper, W. Klootwijk, and T. J. Visser Substitution of Cysteine for Selenocysteine in the Catalytic Center of Type III Iodothyronine Deiodinase Reduces Catalytic Efficiency and Alters Substrate Preference Endocrinology, June 1, 2003; 144(6): 2505 - 2513. [Abstract] [Full Text] [PDF]
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), D2 A131C (
) or D2 A131S (
) enzymes. A, Inhibition of T4 (2 nM) deiodination by wt D2, D2 A131C or D2 A131S enzymes by PTU in the presence of 0.2 mM DTT, but in the presence of 20 mM DTT the wt D2, D2 A131C or D2 A131S enzymes are insensitive to PTU. B, Inhibition of T4 (2 nM) deiodination by wt D2, D2 A131C or D2 A131S enzymes by GTG. C, Inhibition of T4 (2 nM) deiodination by wt D2, D2 A131C, or D2 A131S enzymes by BrAcT4.
