Thyroid hormone transport by the human monocarboxylate transporter 8 and its rate-limiting role in intracellular metabolism.

Cellular entry of thyroid hormone is mediated by plasma membrane transporters. We have identified rat monocarboxylate transporter 8 (MCT8) as an active and specific thyroid hormone transporter. The MCT8 gene is located on the X-chromosome. The physiological relevance of MCT8 has been demonstrated by the identification of hemizygous mutations in this gene in males with severe psychomotor retardation and elevated serum T(3) levels. We have characterized human (h) MCT8 by analysis of iodothyronine uptake and metabolism in cell lines transiently transfected with hMCT8 cDNA alone or together with cDNA coding for iodothyronine deiodinase D1, D2, or D3. MCT8 mRNA was detected by RT-PCR in a number of human cell lines as well as in COS1 cells but was low to undetectable in other cell lines, including JEG3 cells. MCT8 protein was not detected in nontransfected cell lines tested by immunoblotting using a polyclonal C-terminal hMCT8 antibody but was detectable in transfected cells at the expected size (61 kDa). Transfection of COS1 and JEG3 cells with hMCT8 cDNA resulted in 2- to 3-fold increases in uptake of T(3) and T(4) but little or no increase in rT(3) or 3,3'-diiodothyronine (3,3'-T(2)) uptake. MCT8 expression produced large increases in T(4) metabolism by cotransfected D2 or D3, T(3) metabolism by D3, rT(3) metabolism by D1 or D2, and 3,3'-T(2) metabolism by D3. Affinity labeling of hMCT8 protein was observed after incubation of intact transfected cells with N-bromoacetyl-[(125)I]T(3). hMCT8 also facilitated affinity labeling of cotransfected D1 by bromoacetyl-T(3). Our findings indicate that hMCT8 mediates plasma membrane transport of iodothyronines, thus increasing their intracellular availability.

Regulation of type III iodothyronine deiodinase expression in human cell lines.

Type I iodothyronine deiodinase (D1) and type II iodothyronine deiodinase (D2) catalyze the activation of the prohormone T4 to the active hormone T3; type III iodothyronine deiodinase (D3) catalyzes the inactivation of T4 and T3. D3 is highly expressed in brain, placenta, pregnant uterus, and fetal tissues and plays an important role in regulating thyroid hormone bioavailability during fetal development. We examined the activity of the different deiodinases in human cell lines and investigated the regulation of D3 activity and mRNA expression in these cell lines, as well as its possible coexpression with neighboring genes Dlk1 and Dio3os, which may also be especially important during development. D1 activity and mRNA were only found in HepG2 hepatocarcinoma cells, and D2 activity was observed in none of the cell lines. D3 activity and mRNA was found in ECC-1 endometrium carcinoma cells, MCF-7 mammacarcinoma cells, WRL-68 embryonic liver cells, and SH-SY5Y neuroblastoma cells, but not in the HepG2 hepatocarcinoma cell line or in any choriocarcinoma or astrocytoma cell line. We demonstrated that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate increased D3 activity 2- to 9-fold in ECC-1, MCF-7, WRL-68, and SH-SY5Y cells. Estradiol increased D3 activity 3-fold in ECC-1, but not in any other cells. Dexamethasone decreased D3 activity in WRL-68 cells only in the absence of fetal calf serum. Incubation with retinoids increased D3 activity 2- to 3-fold in ECC-1, WRL-68, and MCF-7 cells but decreased D3 activity in SH-SY5Y cells. D3 expression in the different cells was not affected by cAMP or thyroid hormone. Interestingly, D3 mRNA expression in the different cell lines strongly correlated with Dio3os mRNA expression and in a large set of neuroblastoma cell lines also with Dlk1 expression. In conclusion, we identified different human D3-expressing cell lines, in which the regulation of D3 expression is cell type-specific. Our data suggest that estradiol may be one of the factors contributing to the induction of D3 activity in the pregnant uterus and that in addition to gene-specific regulatory elements, more distant common regulatory elements also may be involved in the regulation of D3 expression.

Deiodinase activity is present in Xenopus laevis during early embryogenesis.

Thyroid hormones orchestrate amphibian metamorphosis. The type 2 and type 3 deiodinases make vital contributions to this process by controlling levels of the thyroid hormones T(4) and T(3) available to different tissues. Because the tadpole thyroid gland is not functional until stage NF44, it has been widely assumed that thyroid signaling is absent during amphibian early development, thyroid hormone only becoming a major regulator during premetamorphic stages. Similarly, in mammals, thyroid function is known to be essential to neuronal development, especially during the perinatal stages, but again little is known about early stages of development. Here we demonstrate that key elements of thyroid hormone signaling are present during early development of Xenopus. In particular, we find functional thyroid hormone-activating deiodinases and significant levels of their substrates, T(4) and T(3), during early embryogenesis. Furthermore, we have further characterized a recently identified deiodinase in amphibians, homologous to mammalian type 1 deiodinase (D1). This enzyme is expressed in marked, spatially defined patterns during embryogenesis. The patterns of expression of type 1 deiodinase are distinct from those of type 2 and type 3 deiodinases. Deiodinase expression is found in neurogenic areas from stage NF30 onward, both in the central and peripheral nervous systems. We conclude that both activating and inactivating deiodinases show dynamic patterns of expression during early embryogenesis in amphibians, particularly in neurogenic areas. These findings suggest that thyroid hormone signaling is a key component of early neuronal development in vertebrates.

Characterization of recombinant Xenopus laevis type I iodothyronine deiodinase: substitution of a proline residue in the catalytic center by serine (Pro132Ser) restores sensitivity to 6-propyl-2-thiouracil.

In frogs such as Rana and Xenopus, metamorphosis does not occur in the absence of a functional thyroid gland. Previous studies indicated that coordinated development in frogs requires tissue and stage-dependent type II and type III iodothyronine deiodinase expression patterns to obtain requisite levels of intracellular T(3) in tissues at the appropriate stages of metamorphosis. No type I iodothyronine deiodinase (D1), defined as T(4) or reverse T(3) (rT3) outer-ring deiodinase (ORD) activity with Michaelis constant (K(m)) values in the micromolar range and sensitivity to 6-propyl-2-thiouracil (6-PTU), could be detected in tadpoles so far. We obtained a X. laevis D1 cDNA clone from brain tissue. The complete sequence of this clone (1.1 kb, including poly A tail) encodes an ORF of 252 amino acid residues with high homology to other vertebrate D1 enzymes. The core catalytic center includes a UGA-encoded selenocysteine residue, and the 3' untranslated region (about 300 nt) contains a selenocysteine insertion sequence element. Transfection of cells with an expression vector containing the full-length cDNA resulted in generation of significant deiodinase activity in the homogenates. The enzyme displayed ORD activity with T(4) (K(m) 0.5 microm) and rT3 (K(m) 0.5 microm) and inner-ring deiodinase activity with T(4) (K(m) 0.4 microm). Recombinant Xenopus D1 was essentially insensitive to inhibition by 6-PTU (IC(50) > 1 mm) but was sensitive to gold thioglucose (IC(50) 0.1 mum) and iodoacetate (IC(50) 10 microm). Because the residue 2 positions downstream from the selenocysteine is Pro in Xenopus D1 but Ser in all cloned PTU-sensitive D1 enzymes, we prepared the Pro132Ser mutant of Xenopus D1. The mutant enzyme showed strongly increased ORD activity with T(4) and rT3 (K(m) about 4 microm) and was highly sensitive to 6-PTU (IC(50) 2 microm). Little native D1 activity could be detected in Xenopus liver, kidney, brain, and gut, but significant D1 mRNA expression was observed in juvenile brain and adult liver and kidney. These results indicate the existence of a 6-PTU-insensitive D1 enzyme in X. laevis tissues, but its role during tadpole metamorphosis remains to be defined.

Biochemical mechanisms of thyroid hormone deiodination.

Deiodination is the foremost pathway of thyroid hormone metabolism not only in quantitative terms but also because thyroxine (T(4)) is activated by outer ring deiodination (ORD) to 3,3',5-triiodothyronine (T(3)), whereas both T(4) and T(3) are inactivated by inner ring deiodination (IRD) to 3,3',5-triiodothyronine and 3,3'-diiodothyronine, respectively. These reactions are catalyzed by three iodothyronine deiodinases, D1-3. Although they are homologous selenoproteins, they differ in important respects such as catalysis of ORD and/or IRD, deiodination of sulfated iodothyronines, inhibition by the thyrostatic drug propylthiouracil, and regulation during fetal and neonatal development, by thyroid state, and during illness. In this review we will briefly discuss recent developments in these different areas. These have resulted in the emerging view that the biological activity of thyroid hormone is regulated locally by tissue-specific regulation of the different deiodinases.

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast.

The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone thyroxine (T4) by the hepatic selenoenzyme type I iodothyronine deiodinase (D1), i.e. by outer ring deiodination (ORD) to the active hormone triiodothyronine (T3) or by inner ring deiodination (IRD) to the inactive metabolite reverse T3 (rT3). Since D1 is a membrane-bound protein with an N-terminal membrane-spanning domain, the enzyme is very difficult to purify in an active state. This study was undertaken in order to develop a heterologous (over)-expression system that would eventually allow the production of large amounts of purified active D1 protein. We have expressed a mutant rat D1 protein, in which the selenocysteine residue in the core catalytic center was replaced by cysteine (D1 Cys) in yeast cells (Saccharomyces cerevisiae). After yeast cell fractionation, kinetic analysis was performed with dithiothreitol as reducing cofactor. ORD activity was associated with membrane fractions, while no activity could be detected in the cytosolic fraction. The D1 Cys protein displayed a tenfold increase in Km (2 microM) for rT3 as compared with native D1 protein in rat liver microsomes. The D1 protein content is about 65 pmol/mg microsomal protein, as compared with about 3 pmol/mg in rat liver microsomal fraction. SDS-PAGE analysis of N-bromoacetyl-[125I]T3 affinity-labeled D1 protein showed several labeled protein isoforms with apparent molecular masses between 27 and 32 kDa. Immunoblot analysis with a specific D1 antiserum confirmed the observed D1 protein heterogeneity. Site-directed mutagenesis of several potential N-linked glycosylation sites, phosphorylation sites and a unique myristoylation site established that D1 heterogeneity is not caused by N-linked glycosylation, but probably by a combination of O-linked glycosylation and phosphorylation. Deletion of the endoplasmic reticulum (ER)-signal sequence and the membrane-spanning domain (amino acid residue 2-35), did not result in the production of a soluble D1 enzyme. Although this mutated D1 protein is inactive, the fact that it is still membrane bound indicates the existence of additional membrane attachment site(s) or membrane-spanning domains. Overall, our studies indicate that yeast cells provide a useful system for the expression of relatively high levels of D1 protein which could be used for further structure-function analysis.

A new polymorphism in the type II deiodinase gene is associated with circulating thyroid hormone parameters.

Type II deiodinase (D2) is important in the regulation of local thyroid hormone bioactivity in certain tissues. D2 in skeletal muscle may also play a role in serum triiodothyronine (T(3)) production. In this study, we identified a polymorphism in the 5'-UTR of the D2 gene (D2-ORFa-Gly3Asp). We investigated the association of D2-ORFa-Gly3Asp, and of the previously identified D2-Thr92Ala polymorphism, with serum iodothyronine levels. D2-ORFa-Gly3Asp was identified by sequencing the 5'-UTR of 15 randomly selected individuals. Genotypes for D2-ORFa-Gly3Asp were determined in 156 healthy blood donors (age 46.3 +/- 12.2 yr) and 349 ambulant elderly men (age 77.7 +/- 3.5 yr) and related to serum iodothyronine and TSH levels. D2-ORFa-Asp(3) had an allele frequency of 33.9% in blood bank donors and was associated with serum thyroxine (T(4); Gly/Gly vs. Gly/Asp vs. Asp/Asp = 7.06 +/- 0.14 vs. 6.74 +/- 0.15 vs. 6.29 +/- 0.27 microg/dl, P = 0.01), free T(4) (1.22 +/- 0.02 vs. 1.16 +/- 0.02 vs. 1.06 +/- 0.04 ng/dl, P = 0.001), reverse T(3) (P = 0.01), and T(3)/T(4) ratio (P = 0.002) in a dose-dependent manner, but not with serum T(3) (P = 0.59). In elderly men, D2-ORFa-Asp(3) had a similar frequency but was not associated with serum iodothyronine levels. This new polymorphism in the 5'-UTR of D2 is associated with iodothyronine levels in blood donors but not in elderly men. We hypothesize that this might be explained by the decline in skeletal muscle size during aging, resulting in a relative decrease in the contribution of D2 to serum T(3) production.

Association between mutations in a thyroid hormone transporter and severe X-linked psychomotor retardation.

Monocarboxylate transporter 8 (MCT8) is a thyroid hormone transporter, the gene of which is located on the X chromosome. We tested whether mutations in MCT8 cause severe psychomotor retardation and high serum triiodothyronine (T3) concentrations in five unrelated young boys. The coding sequence of MCT8 was analysed by PCR and direct sequencing of its six exons. In two patients, gene deletions of 2.4 kb and 24 kb were recorded and in three patients missense mutations Ala150Val, Arg171 stop, and Leu397Pro were identified. We suggest that this novel syndrome of X-linked psychomotor retardation is due to a defect in T3 entry into neurons through MCT8, resulting in impaired T3 action and metabolism.

Characteristics and thyroid state-dependent regulation of iodothyronine deiodinases in pigs.

Three iodothyronine deiodinases (D1, D2, and D3) regulate local and systemic availability of thyroid hormone. D1 and D2 activate the prohormone T4 to the thyromimetic T3, and D3 inactivates T4 and T3 to rT3 and 3,3'-diiodothyronine, respectively. The expression of the three deiodinases is tightly regulated with regard to developmental stage and cell type to provide fine tuning of T3 supply to target cells. Most studies regarding distribution and regulation of deiodinases have been carried out in rodents. However, in different respects, rodents do not seem to be the optimal experimental model for human thyroid hormone physiology. For instance, D2 expression has been observed in human thyroid and skeletal muscle but not in these tissues in rodents. In this study, we have explored the pig as an alternative model. Porcine D1, D2, and D3 were cloned by RT-PCR, and their catalytic properties were shown to be virtually identical to those reported for human and rodent deiodinases. The tissue distribution of deiodinases was studied in normal pigs and in pigs made hypothyroid by methimazole treatment or in pigs made hyperthyroid by T4 treatment. D1 activity in liver and kidney was increased in T4-treated pigs. D2 activities in cerebrum and pituitary were decreased after T4 treatment and strongly increased after methimazole treatment. Remarkably, D2 activity in thyroid and skeletal muscle was induced in hypothyroid pigs. Significant expression of D3 was observed in cerebrum and was positively regulated by thyroid state. In conclusion, the pig appears to be a valuable model for human thyroid hormone physiology. The expression of D2 activity in thyroid and skeletal muscle is of particular interest for studies on the importance of this enzyme in (hypothyroid) humans.

An ascidian homolog of vertebrate iodothyronine deiodinases.

In all classes of vertebrates, the deiodination of the prohormone T(4) to T(3) represents an essential activation step in thyroid hormone action. The possible presence of iodothyronine deiodinase activity in protochordates has been demonstrated in vivo. Recent molecular cloning of the genomes and transcripts of several ascidian species allows further investigation into thyroid-related processes in ascidians. A cDNA clone from Halocynthia roretzi (hrDx) was found to have significant homology (30% amino acid identity) with the iodothyronine deiodinase gene sequences from vertebrates, including the presence of an in-frame UGA codon that might encode a selenocysteine (SeC) in the active site. Because it was not certain that the 3' untranslated region (UTR) contained a SeC insertion sequence (SECIS) element essential for SeC incorporation, a chimeric expression vector of the hrDx coding sequence and the rat deiodinase SECIS element was produced, as well as an expression vector containing the intact hrDx cDNA. COS, CHO, and HEK cells were transfected with these vectors, and deiodinase activity was measured in cell homogenates. Outer-ring deiodinase activity was detected using both T(4) and reverse T(3) as substrates, and activity was enhanced by the presence of the reductive cofactor dithiothreitol. The enzyme activity was optimal during incubation between 20 and 30 C (pH 6-7) and was strongly inhibited by gold-thioglucose. The Halocynthia deiodinase appears to be a high Michaelis-Menten constant (K(m)) enzyme (K(m) reverse T(3), 2 microM; and K(m) T(4), 4 microM). Deiodinase activity was completely lost upon the substitution of the SeC residue in the putative catalytic center by either cysteine or alanine. Transfection of the full-length hrDx cDNA produced deiodinase activity confirming the presence of a SECIS element in the 3'UTR, as revealed by the SECISearch program. In conclusion, our results show, for the first time, the existence of an ascidian iodothyronine outer-ring deiodinase. This raises the hypothesis that, in protochordates, the prohormone T(4) is activated by enzymatic outer-ring deiodination to T(3).

Molecular basis for the substrate selectivity of cat type I iodothyronine deiodinase.

The type I iodothyronine deiodinase (D1) catalyzes the activation of T4 to T3 as well as the degradation of T3 (rT3) and sulfated iodothyronines. A comparison of the catalytic activities of D1 in liver microsomal preparations from several species revealed a remarkable difference between cat D1 on one hand and rat/human D1 on the other hand. The Michaelis constant (Km) of cat D1 for rT3 (11 microm) is 30-fold higher than that of rat and human D1 (0.2-0.5 microm). Deiodination of rT3 by cat D1 is facilitated by sulfation [maximal velocity (Vmax)/Km rT3 = 3 and Vmax/Km rT3S = 81]. To understand the molecular basis for the difference in substrate interaction the cat D1 cDNA was cloned, and the deduced amino acid sequence was compared with rat/human D1 protein. In the region between amino acid residues 40 and 70 of cat D1, various differences with rat/human D1 are concentrated. By site-directed mutagenesis of cat D1 it was found that a combination of mutations was necessary to improve the deiodination of rT3 by cat D1 enzyme. For efficient rT3 deiodination, a Phe at position 65 and the insertion of the Thr-Gly-Met-Thr-Arg48-52 sequence as well as the amino acids Gly and Glu at position 45-46 are essential. Either of these changes alone resulted in only a limited improvement of rT3 deiodination. At the same time the combination of the described mutations did not affect the already quite efficient outer ring deiodination of rT3S nor the inner ring deiodination of T3S, whereas each of the described changes alone did affect rT3S deiodination. Our findings suggest great flexibility of the active site in D1 that adapts to its various substrates. The active site of wild-type cat D1 is less flexible than the active site of rat/human D1 and favors sulfated iodothyronines.

Polymorphisms in thyroid hormone pathway genes are associated with plasma TSH and iodothyronine levels in healthy subjects.

Single nucleotide polymorphisms (SNPs) in genes involved in thyroid hormone metabolism may affect thyroid hormone bioactivity. We investigated the occurrence and possible effects of SNPs in the deiodinases (D1-D3), the TSH receptor (TSHR), and the T(3) receptor beta (TR beta) genes. SNPs were identified in public databases or by sequencing of genomic DNA from 15 randomly selected subjects (30 alleles). Genotypes for the identified SNPs were determined in 156 healthy blood donors and related to plasma T(4), free T(4), T(3), rT(3), and TSH levels. Eight SNPs of interest were identified, four of which had not yet been published. Three are located in the 3'-untranslated region: D1a-C/T (allele frequencies, C = 66%, T = 34%), D1b-A/G (A = 89.7%, G = 10.3%), and D3-T/G (T = 85.5%, G = 14.2%). Four are missense SNPs: D2-A/G (Thr92Ala, Thr = 61.2%, Ala = 38.8%), TSHRa-G/C (Asp36His, Asp = 99.4%, His = 0.6%), TSHRb-C/A (Pro52Thr, Pro = 94.2%, Thr = 5.8%), and TSHRc-C/G (Asp727Glu, Asp = 90.7%, Glu = 9.3%). One is a silent SNP: TR beta-T/C (T = 96.8%, C = 3.2%). D1a-T was associated in a dose-dependent manner with a higher plasma rT(3) [CC, 0.29 +/- 0.01; CT, 0.32 +/- 0.01; and TT, 0.34 +/- 0.02 nmol/liter (mean +/- SE); P = 0.017], a higher plasma rT(3)/T(4) (P = 0.01), and a lower T(3)/rT(3) (P = 0.003) ratio. The D1b-G allele was associated with lower plasma rT(3)/T(4) (P = 0.024) and with higher T(3)/rT(3) (P = 0.08) ratios. TSHRc-G was associated with a lower plasma TSH (CC, 1.38 +/- 0.07, vs. GC, 1.06 +/- 0.14 mU/liter; P = 0.04), and with lower plasma TSH/free T(4) (P = 0.06), TSH/T(3) (P = 0.06), and TSH/T(4) (P = 0.08) ratios. No associations with TSH and iodothyronine levels were found for the other SNPs. We have analyzed eight SNPs in five thyroid hormone pathway genes and found significant associations of three SNPs in two genes (D1, TSHR) with plasma TSH or iodothyronine levels in a normal population.

Substitution of cysteine for selenocysteine in the catalytic center of type III iodothyronine deiodinase reduces catalytic efficiency and alters substrate preference.

Human type III iodothyronine deiodinase (D3) catalyzes the conversion of T(4) to rT(3) and of T(3) to 3, 3'-diiodothyronine (T2) by inner-ring deiodination. Like types I and II iodothyronine deiodinases, D3 protein contains selenocysteine (SeC) in the highly conserved core catalytic center at amino acid position 144. To evaluate the contribution of SeC144 to the catalytic properties of D3 enzyme, we generated mutants in which cysteine (D3Cys) or alanine (D3Ala) replaces SeC144 (D3wt). COS cells were transfected with expression vectors encoding D3wt, D3Cys, or D3Ala protein. Kinetic analysis was performed on homogenates with dithiothreitol as reducing cofactor. The Michaelis constant of T(3) was 5-fold higher for D3Cys than for D3wt protein. In contrast, the Michaelis constant of T(4) increased 100-fold. The D3Ala protein was enzymatically inactive. Semiquantitative immunoblotting of homogenates with a D3 antiserum revealed that about 50-fold higher amounts of D3Cys and D3Ala protein are expressed relative to D3wt protein. The relative substrate turnover number of D3Cys is 2-fold reduced for T(3) and 6-fold reduced for T(4) deiodination, compared with D3wt enzyme. Studies in intact COS cells expressing D3wt or D3Cys showed that the D3Cys enzyme is also active under in situ conditions. In conclusion, the SeC residue in the catalytic center of D3 is essential for efficient inner-ring deiodination of T(3) and in particular T(4) at physiological substrate concentrations.

Induction of thyroid hormone-degrading deiodinase in cardiac hypertrophy and failure.

The similarities between the changes in cardiac gene expression in pathological ventricular hypertrophy and hypothyroidism suggest a role of impaired cardiac thyroid hormone (TH) action in the development of contractile dysfunction during chronic cardiac pressure overload. Here we studied the possible involvement of altered cardiac TH metabolism using a rat model of right-ventricular (RV) hypertrophy induced by pressure-overload. Pathological RV hypertrophy was indicated by decreased mRNA levels of sarcoplasmic reticulum(SR) Ca2-ATPase type 2a (SERCA2a) and myosin heavy chain a (MHCalpha), and increased levels of MHCbeta mRNA. Enzyme activity of type HI deiodinase (D3), which converts T4 and T3 to the inactive compounds rT3 and 3,3'-T2, respectively, was identified in ventricular tissue. This activity was stimulated up to five fold in hypertrophic RV, but remained unaltered in the non-hypertrophic left ventricle (LV). A low level of type Ideiodinase activity was also detected, which decreased significantly in both RV and LV. Stimulation of RV D3 activity was significantly higher in those animals in which hypertrophy progressed to heart failure, compared to animals that developed compensatory hypertrophy. The induction of a cardiac TR-degrading deiodinase maybe expected to result in reduced cellular levels of T3 and thereby contribute to a local hypothyroid state in the hypertrophic and, particularly, in the failing ventricle.

Substitution of cysteine for a conserved alanine residue in the catalytic center of type II iodothyronine deiodinase alters interaction with reducing cofactor.

UNLABELLED: Human type II iodothyronine deiodinase (D2) catalyzes the activation of T(4) to T(3). The D2 enzyme, like the type I (D1) and type III (D3) deiodinases, contains a selenocysteine (SeC) residue (residue 133 in D2) in the highly conserved catalytic center. Remarkably, all of the D2 proteins cloned so far have an alanine two residue-amino terminal to the SeC, whereas all D1 and D3 proteins contain a cysteine at this position. A cysteine 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. We have investigated whether D2 mutants with a cysteine (A131C) or serine (A131S) two-residue amino terminal to the SeC are enzymatically active and have characterized these mutants with regard to substrate affinity, reducing cofactor interaction and inhibitor profile. 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 T(4) (5 nM) and reverse T(3) (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 (IC(50)) > 2 mM] in the presence of 20 mM DTT, but when tested in the presence of 0.2 mM DTT the IC(50) 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 T(4) resulted in the saturation of [(125)I]T(4) deiodination, as reflected in a decrease of [(125)I]T(3) release into the medium. Saturation first appeared at medium T(4) 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.