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Thyroid Division, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: P. Reed Larsen, M.D., Thyroid Division, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115. E-mail: larsen{at}rascal.med.harvard.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ORD catalyzed by D1 requires the presence of a reduced thiol cosubstrate and exhibits ping-pong bisubstrate kinetics (12). Biochemical studies suggest that an iodothyronine substrate initially reacts with the enzyme, yielding a deiodinated product and an enzyme-iodide complex. Subsequent binding with a thiol cosubstrate releases an iodide ion and regenerates active enzyme. Inhibition of type 1 deiodination with the thiourea drug 6-n-propylthiouracil (PTU) is uncompetitive with respect to iodothyronine and competitive with respect to thiol cosubstrate. Current evidence suggests that PTU reacts with the enzyme-iodide complex through formation of a seleno-sulfur bond and prevents interaction with the thiol cosubstrate (12, 13).
Sequence analysis and mutational studies have identified amino acid
residues that are important to the function of D1 (4, 13, 14, 15). The
presence of selenocysteine accounts for many of the biochemical
properties that characterize D1 ORD, including high catalytic
efficiency, substrate affinity, and sensitivity to inhibition by PTU
and gold thioglucose (13, 16). An active site cysteine is located two
residues amino-terminal to the selenocysteine in all deduced sequences
of D1 and D3 (Fig. 1
) (2, 3, 4, 5, 8, 9, 10). Type
2 deiodinases, however, contain an alanine at this position. A cysteine
residue could assist in enzymatic action by providing a nucleophilic
sulfur atom or by participating in catalytically relevant disulfide
bond formation (6, 7). The present studies were performed to elucidate
the functional role of the active site cysteine in D1. We examined the
effects of a C124A mutation on substrate affinity and enzymatic
activity of rat type 1 deiodinase for both ORD and IRD. The results
indicate that Cys124 is involved in thiol cosubstrate-D1
interactions, providing further insight into the catalytic mechanism of
iodothyronine deiodination catalyzed by D1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of the D1 mutant
A complementary DNA encoding rat type 1 deiodinase was
previously subcloned into the mammalian expression vector D10 (D1-D10)
(18). A rat D1 PCR fragment containing a C124A mutation was generated
via oligonucleotide-directed mutagenesis as previously described (19).
The C124A rat D1 mutant was created by using a 5'-PstI site
and a 3'-AccI site that were found in the same relative
positions in both the mutation-containing PCR fragment and the D1-D10
construct. The entire coding region of the C124A construct was
sequenced to confirm that no other mutations were present.
DNA transfections
Transfection of 293-HEK cells by calcium phosphate precipitation
was described previously (20). Two days after transfection, cells were
harvested and sonicated in 0.1 M potassium phosphate, 1
mM EDTA, pH 6.9 (PE buffer), containing 25 mM
dithiothreitol (DTT). Transfection efficiencies were monitored by assay
of human GH in the medium derived by cotransfecting a constitutive
thymidine kinase promoter-directed human GH-expressing plasmid, pTKGH
(20). The protein concentration in cell sonicates was measured by
Bio-Rad (Richmond, CA) determination, using human 
-globulin as a
standard.
BrAc[125I]T3
affinity labeling and enzyme quantitation
BrAc[125I]T3 was synthesized from
N-bromoacetylchloride and [125I]T3
as described previously (21). The product was purified on LH-20
Sephadex by elution with ethanol. Purity was verified by TLC in ethyl
acetate-glacial acetic acid (4:1). Affinity labeling reactions were
performed by incubating 50 µg cell sonicate with PE, 20 fmol (0.1
µCi) BrAc[125I]T3, and varying
concentrations of unlabeled BrAcT3 in a 100-µl final
volume. Reactions proceeded at room temperature for 20 min and were
terminated by the addition of gel loading buffer containing SDS and
ß-mercaptoethanol, followed by boiling for 5 min. Samples were
analyzed by electrophoresis on 12.5% minigels, followed by
autoradiography. Densitometric quantitation of the autoradiographs was
performed using a Molecular Dynamics computing densitometer (Sunnyvale,
CA). The relative quantity of enzyme was determined by plotting the
magnitude of specifically bound protein against the inverse of the
unlabeled BrAcT3 concentration. This is derived by
rearranging the formula P + nA + nA1 
PAn +
PA1n, where P is the protein concentration, A is the
covalent substrate, 1 represents radiolabeled substrate, and n is the
number of binding sites, to the equation (specifically bound
label/total label) = (nP/A0), where A0 is the
initial concentration of covalent substrate.
Synthesis of T3 sulfate
[125I]T3S was synthesized from
T3 and chlorosulfonic acid as previously described (22). A
solution containing 200 fmol to 1 µmol
[125I]T3 or unlabeled T3 in 1
M ammonium hydroxide in ethanol was dried under a nitrogen
stream. A mixture of 200 µl 15 M ClSO3H was
slowly added to 800 µl dimethylformamide at 0 C. Subsequently, 200
µl ClSO3H-dimethylformamide solution were added to the
T3 residue at 0 C. Reactions were continued overnight at
room temperature, and products were purified on an LH-20 Sephadex
column by elution with H2O. Purity was determined by TLC in
ethyl acetate-glacial acetic acid (4:1) solvent.
Deiodinase assays
ORD was assayed by the release of 125I-
from outer ring-labeled rT3. Reactions contained 0.9–3.0
µg sonicate protein, varying concentrations of DTT (0.4–10
mM) or glutathione (GSH; 5 mM), 100,000 cpm
[125I]rT3 (DuPont-New England Nuclear,
Boston, MA), varying concentrations of PTU (0–1.0 mM), and
varying concentrations of unlabeled rT3 (20–200
nM for DTT cosubstrate experiments; 0.25–1 nM
for GSH cosubstrate experiments) in PE buffer (13). After incubation at
37 C for 1 h, reactions were terminated by the addition of 200
µl horse serum and 100 µl trichloroacetic acid. Reaction mixtures
were centrifuged for 15 min at 15,000 x g, and
supernatants were counted for 125I-. All
reactions were performed in duplicate, and experiments for wild-type
and C124A enzymes were performed simultaneously.
IRD assays were performed by measuring 125I- release from outer ring-labeled T3 sulfate as described previously (23). Reactions contained 9 µg sonicate protein, 10 mM DTT, 100,000 cpm [125I]T3S and varying concentrations of unlabeled T3S (0.4–6 µM) in PE buffer. After incubation at 37 C for 1 h, reactions were terminated by the addition of 200 µl horse serum and 100 µl trichloroacetic acid. Reaction mixtures were centrifuged for 15 min at 15,000 x g, and supernatants were counted for 125I-. All reactions were performed in duplicate, and experiments for wild-type and C124A enzymes were performed simultaneously.
To confirm that IRD of T3 sulfate (T3S) to 3,3'-diiodothyronine sulfate (T2S) was the rate-limiting step for both the C124A mutant and the wild-type D1, we characterized the reaction products at different time points by differential chromatography on LH-20 Sephadex. Preliminary studies confirmed that I-, T2, and T3 can be separated by application of the products to an LH-20 column in aqueous medium (23). Iodide was not retarded, and T2, but not T3, was eluted by 20% ethanol. T3 was not eluted until the ethanol concentration reached 50%. T3S and T2S were not significantly retarded on LH-20. The T3S deiodination reactions at 100–200 nM T3S with both wild-type and C124A mutant were terminated by the addition of 1 ml 1 N HCl, followed by incubation at 80 C for 60 min to hydrolyze the sulfated iodothyronines (23). The samples were then neutralized with 1 N NaOH and applied to LH-20 columns. Appropriate control studies showed that acid hydrolysis did not cause deiodination of T3 or T2. After 15, 30, 45, and 60 min with both wild-type and C124A mutant D1 enzymes, the only labeled products were 125I- and [125I]T3 (at early time intervals), with less than 0.5% of the T3S added appearing as [125I]T2. Thus, metabolism of T3S proceeds through IRD to T2S, followed by rapid ORD of this product. IRD of T3S is the rate-determining step, and direct ORD of T3S does not occur (23). Accordingly, 125I- release is a proxy for IRD of T3S (23).
Kinetic analysis
Reaction conditions were chosen so that less than 30% of the
substrate was consumed during the enzyme-catalyzed reaction. Average
values of closely agreeing duplicate samples (±10%) were used for
Lineweaver-Burk analysis, and plots were generated by unweighted least
squares analysis. A minimum of three separate experiments were
performed to generate each kinetic constant. Intercept replots were
also fitted by linear regression. All kinetic parameters were compared
by unpaired Student’s t test, assuming equal variance in
the underlying populations. P < 0.05 was considered
significant. Regression analysis and t tests were performed
with Microsoft Excel software (Microsoft, Redmond, WA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The concentration of
in vitro expressed wild-type D1 was 1.1 ± 0.035-fold
greater than that of C124A (n = 3). This finding is consistent
with GH assays that demonstrate similar transfection efficiencies for
wild-type and C124A enzymes (data not shown). As maximal velocity
(Vmax) is dependent on the enzyme concentration, all
estimates of the apparent Vmax in subsequently described
kinetic experiments were corrected for the 10% difference in wild-type
and C124A concentrations.
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, and
typical Lineweaver-Burk plots of rT3 ORD and
T3S IRD are shown in Fig. 3, A and B. The Lineweaver-Burk plot for the C124A mutant demonstrated an
upward parallel shift relative to the wild-type enzyme. For ORD, the
C124A mutant exhibited significantly lower values for apparent
Vmax and Km compared with those of
the wild-type enzyme, whereas the
Vmax/Km ratio for both enzymes was
the same (Table 1). Similarly, the apparent Vmax and
Km values for IRD were significantly lower for the C124A
enzyme, whereas there was no difference in the
Vmax/Km ratio. The catalytic turnover
constant (kcat)/Km ratio is an apparent second
order rate constant that represents the catalytic properties of free
enzyme (26). At equal enzyme concentrations, the
Vmax/Km ratios of wild-type and
mutant enzymes are proxies for kcat/Km.
Replacement of Cys124 by Ala reduced the apparent
Vmax and Km for both ORD and IRD, but
had no effect on the catalytic efficiency of free enzyme for either
deiodination pathway.
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and Fig. 3
suggest that the effective cosubstrate concentration for the C124A
mutant is reduced relative to that of the wild-type enzyme. To study
the possibility that Cys124 plays a role in cosubstrate
binding, rT3 ORD was studied at several concentrations of
DTT. Representative experiments for the wild-type and C124A enzymes are
shown in Fig. 4, A and B. Studies were
performed at nonsaturating concentrations of DTT (0.4–5
mM), and the Lineweaver-Burk plots were derived for each
DTT concentration. A replot of the reciprocal of the apparent
Vmax against the reciprocal of the DTT concentration
yielded the limiting Michaelis constant for DTT
(KmDTT) and the Vmax (Table 2) (28). Similar analysis of
Lineweaver-Burk plots of the same data at different DTT but fixed
rT3 concentrations provided the limiting Michaelis constant
for rT3 (KmrT3). The
KmDTT of the C124A mutant was 14-fold greater
than that of the wild-type D1 (P < 0.05). No
significant differences in KmrT3 or
Vmax between the wild-type and mutant D1 enzymes were found
in these experiments.
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. A replot of 1/Vm against PTU
concentration yielded the KiPTU (Table 2). The
C124A mutant was uncompetitively inhibited by PTU with respect to
rT3. The KiPTU of the C124A mutant
was 2.3-fold greater than that of the wild-type D1 (P
< 0.05).
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). The apparent
Km of the mutant for rT3 is lower
than that for the wild type, whereas no significant differences were
detected in apparent Vmax and
Vmax/Km (Table 3). ORD of rT3 mediated by
the C124A mutant was again displaced in an upward, parallel fashion
relative to that catalyzed by the wild-type enzyme. These results
suggest that the C124A mutation has similar effects on enzyme catalysis
in the presence of either mono- or dithiols.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The specific preference of various iodothyronines for ORD or IRD may be determined by the conformation of bound substrate and the relative proximities of inner vs. outer ring iodide atoms to active site residues. For example, recent work has identified Phe65 in human D1 as important for the interactions of D1 with the monoiodinated, but not the diiodinated, tyrosyl ring of iodothyronines, suggesting that this residue plays a key role in the interaction of rT3 and T2S with D1 (4, 14). Thus, a Phe65Leu substitution (present in the wild-type dog D1) reduces ORD of rT3 and 3,3' T2S, but not IRD of T3S (4, 14). In contrast, the present study demonstrates that optimal ORD of rT3 and IRD of T3S by D1 requires C124. These observations argue that C124 plays an important role in catalysis subsequent to the binding of the iodothyronine substrate.
The present results demonstrate that Cys124 enhances thiol affinity, as reflected in the KmDTT. For both ORD and IRD, Lineweaver-Burk plots for C124A were shifted upward and parallel relative to wild-type D1. Thus, the C124A mutation adversely affects the interaction of DTT with D1. Secondary plot analysis demonstrated a 14-fold increase in KmDTT for the mutant D1 without significant changes in rT3 affinity or Vmax. An increase of this magnitude in KmDTT is predicted by the Michaelis-Menten equation to halve the apparent Vmax in the presence of 10 mM DTT, and experimental data from the present study confirm this expected effect. Double reciprocal plots at varying DTT concentrations yield a set of parallel lines for C124A, implying that the ping-pong mechanism of deiodination is not altered by the mutation.
The values presented in Table 2 for the limiting
Km for rT3 and DTT of the
wild-type enzyme are, respectively, 20- and 10-fold lower than
previously reported values of 460 and 8.1 nM (27). Reported
experiments were performed at initial rate conditions within the first
1–3 min of the deiodination reaction. Our assays examined deiodination
rates after 1 h. Although reaction conditions were designed so
that less than 30% of the initial substrate was deiodinated, the
possibility of product inhibition of the deiodination reaction cannot
be excluded. Trapping of rT3 and DTT in enzyme
intermediate complexes due to product inhibition could explain the
lower values of KmrT3 and
KmDTT than those previously reported. These
observations do not change our conclusions regarding the qualitative
kinetic differences between the wild-type and C124A mutant D1
enzymes.
PTU inhibits D1 activity uncompetitively with respect to iodothyronine substrate and competitively with respect to DTT (12). These observations suggest that an oxidized Se+ enzyme intermediate is generated after deiodination of an iodothyronine substrate, and PTU is thought to bind to this intermediate (29, 30). The present studies demonstrate similar uncompetitive inhibition of the C124A mutant by PTU, providing evidence that a Se+ intermediate is generated even in the absence of an active site cysteine. The loss of the cysteine residue in this position seems to selectively increase KmDTT without changing the basic catalytic features of deiodination by D1.
The present results suggest an important role of C124A consistent with
the Se+ intermediate model (Fig. 7A). A sulfur-seleno bond forms between
C124 and the oxidized Se+ intermediate. Interaction between
a free thiol group of DTT and C124 forms a new disulfide bond and
releases a reduced selenium group. Subsequent DTT intradisulfide bond
formation regenerates the reduced form of Cys124 and
releases oxidized DTT. The net effect of this pathway is the indirect
reduction of Se+ by DTT through interactions with C124.
Indirect reduction of Se+ may be more efficient than direct
reduction by DTT for several reasons. Steric factors may hinder Se-DTT
interactions, and the formation of a mixed Se-S bond may be
thermodynamically unfavorable (35). A disulfide bond is 10 Cal/mol
stronger than a mixed seleno-sulfur bond, and the former is favored at
equilibrium conditions (36). In the proposed model (Fig. 7B), the
absence of Cys124 decreases efficient reduction of the
Se+ intermediate and lowers the apparent regenerative
effect of DTT.
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The active site cysteine is also important in interactions with the
monothiol GSH, as suggested by similar upward parallel displacement of
C124A Lineweaver-Burk plots relative to those of the wild type.
Previous studies suggest that GSH may be a relevant physiological
cosubstrate for D1 (32, 33). GSH is a less potent and less effective
stimulator of type 1 deiodination than DTT. At 5–40 nM
levels of T4 and rT3, physiological GSH
concentrations of 1–5 mM activate low
Km (
20 nM) deiodination in kidney
and liver microsomes. Subsequent work using in vitro
expressed enzyme demonstrated that this GSH-activated, low
Km activity was also mediated by D1 (37). The
present study demonstrates that loss of Cys124 lowers the
apparent Km and Vmax for
rT3 and reduces the potency of GSH, as occurs
with DTT.
Prior studies of GSH-activated deiodination have suggested a ternary complex mechanism rather than the ping-pong kinetics observed in DTT-activated deiodination (33). In contrast to mechanisms proposed for the latter, the ternary complex model requires the initial reduction of an active center seleno-sulfur bond by one molecule of GSH before substrate binding can occur. Subsequent deiodination of the substrate generates a Se+ intermediate, and interaction with a second molecule of GSH regenerates the active center Se-S bond.
A potential explanation for these disparate observations and models may
depend on the potent reducing properties of DTT compared with those of
GSH. DTT may nonspecifically reduce an active center mixed Se-S bond,
obviate the requirement for initial cosubstrate binding to the enzyme,
and consequently exhibit ping-pong kinetics. The present results
suggest that the C124A mutation eliminates the putative seleno-sulfur
bond and the requirement for a ternary complex mechanism. Deiodination
could then occur, as illustrated in Fig. 7B. Loss of the Se-S bond
facilitates rT3 binding and could explain the reduced
apparent KmrT3 of the C124A mutant.
The active site cysteine thus plays an important role in reducing the
Se+ intermediate in both ping-pong and ternary models. Loss
of this residue decreases the efficiency of regenerating the active
enzyme, and this explanation is consistent with the observed reduction
in the apparent Vmax of the mutant D1.
Alternatively, the observed effects of the C124A mutation could be attributed to conformational alterations rather than changes in cosubstrate affinity. Further independent structural information will be required to exclude this possibility. However, it seems unlikely that a structural change in the active site could specifically cause a 14-fold reduction in DTT affinity and a 2-fold reduction in PTU sensitivity without significant effects on substrate affinity, Vm, or the characteristic catalytic features of this reaction.
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Footnotes |
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Received June 2, 1997.
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