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On the mechanism of inhibition of enzymatic halogenation by antithyroid agents
Life Sciences No . 7, pp . 321-325, 1962 " Great Britain .
Pergamon Press Ltd.
Printed in
ON THE MECHANISM OF INHIBITION OF ENZYMATIC HALOGÉNATION BY ANTITHYROID AGENTS David Morris, Horst Eberwein and Lowell P . Hager Biochemistry Division, University of Illinois, Urbana, Illinois (Received 20 June 1962) CHLOROPEROXIDASE preparations oxidize chloride, l-4 iodide and bromide ion with the formation of the corresponding carbon-halogen bond when supplied with a suitable acceptor molecule . 5
This finding draws a close analogy to the formation
of thyroid hormone which is further strengthened by the observation that chloroperoxidase action is inhibited by the classical antithyroid agents such as thiourea and thiouracil .2
The mechanism by which these compounds inhibit enzy-
matic halogenation is not known although several hypotheses have been suggested on the basis of experimental findings arising from studies concerning the biosynthesis of thyroid hormone . The chloroperoxidase system offers several advantages for a study of. the mechanism of inhibition of enzymatic halogenation by these antithyroid agents especially with 1,1-dimethyl-4-chloro-3,5-cyclohexanedione (monochlorodimedone), chloride ion and hydrogen peroxide as substrates (equation 1) .
Firstly, the
product of the enzymatic halogenation reaction, 1,1-dimethyl-4,4-dichloro-3,5-~ cyclohexanedione (dichlorodimedone), can be readily measured by either an optical or radioactive assay .
Secondly, with either chloride or bromide ion as substrate
the kinetics of the halogen reaction are not complicated by the non-enzymatic oxidation of the halogen anion such as occurs with iodide and hydrogen peroxide . ÇI
CI + C1
_
+ Hy02 + H+ ---~-
321
CI
0 0 ~ + ZHZO
(1)
èIECHANISM OF INHIBITI(~i OF ENZYMATIC HALOGÉNATION
322
No .7
A series of experimental findings with the chloroperoxidase reaction prompts us to propose a general mechanism for the inhibition of enzymatic halogenation by thiouracil . z
80
0 W O W W z O C W O O _U
0
500 I
1000
/ MONOCHLORODIMEDONE
1500 (MOLES / LITER)
FIG . 1 Effect of thiouracil concentration on the enzymatic formation of dichlorodimedone at various concentrations of monochlorodimedone . The complete system (~~) contained the components listed in Table 1 at the indicated concentrations of monochlorodimedone . The effect of increasing amounts of thiouracil on the enzymatic chlorination reaction is shown in curves 2, 3 and 4, where thio uracil is present in the following concentrationss ( - ~ 1 x lo'~ M ; (~-~) z x l0'4 M ; (~-~) 4 x l0'4 M .
Q Q
Resu is Table 1 shows the inhibition of dichlorodimedone formation by thiouracil and the reversal of this inhibition by increasing amounts of monochlorodimedone . Figure 1 graphically presents a series of experimental curves resulting from a plot of the reciprocal of the rate of dichlorodimedone formation against the reciprocal of the monochlorodimedone concentration at various levels of
No .7
MECHANISM OF INHIBITION OF ENZYMATIC HALOGÉNATION
323
TABLE 1 Inhibition of Dichlorodimedone Formation by Thiouracil and its Reversal by Monochlorodimedone Monochlorodimedone added
Dichlorodimedone formation
(pmoles)
1.
0 .5
179
2.
2 .5
202
3.
5 .0
214
4"
10 .0
5.
20 .0
194
6.
40 .0
190 194
The complete system conta}qed 500 pmoles of potassium phosphate buffer, pH 2 .75, 50 moles of KC1 (22,400 cpm/mole), 10 umoles of hydrogen peroxide, 0.5 ~9 chloroperoxidase plus the indicated amounts of monochlorodimedone in a total volume of 5 ml . In reactigns containing thioùracil, it was present at a concentration of 2 x 10 - M. The reaction mixtures were incubated at 30 ° for 4 min. Optical assays indicate that the rate of dichlorodimedone formation is constant during this 4 min incubation period . The reaction was stopped by the addition of 1 ml of 7 N sulfuric acid . Dichlorodimedone formation was measured by exhaustive extraction of the reaction mixtures with ether and determination of the radioactivity in the ether phase . Dichlorodimedone was shown to be the product of the reaction by repeated crystallization of the radioactive enzymatic product with synthetic dichlorodimedone . When 106,500 cpm of radioactive product was mixed with 200 mg of synthetic non-radioactive dichlorodimedone, the specific activity (cpm/mg) after successive crystallizations from petroleum ether was 534, 529, 517, 524 . thiouracil .
These curves are typical of those cases where competitive inhibition
is brought about by a competing substrate . 6 The major end-product of this reaction has been identified as uracil by cochromatography with authentic uracil and by a comparison of the radioactive products obtained from C U`- and S35 -labeled thiouracil (Fig . 2) .
Paper chromato
grams show that at least 80 per cent of the C 14-thiouracil is converted to C 14 uracil .
In contrast, when S35 -thiouracil is used as substrate, no radioactivity
is associated with the uracil spot on the paper chromatogram .
Present evidence
indicates that a majority of the sulfur from thiouracil is converted to elemental sulfur . Although a halogen anion (i .e ., chloride, bromide or iodide) is required
324
MECHANISM OF INHIBITION OF ENZYMATIC HALOGÉNATION
~ 0
1
EXPERIMENT
I
S"= THIOURACIL
ô
10 _EXPERIMENT 2
C I4 -THIOURACIL
No .7
MARKERS I
0 o~lalN
I
THIOURACIL
QI
I
0.2
I
0.3
URACIL I
I
I
0.4
O.s
0.8
Rf VALUES
1
L0 s v r
FIG . 2 Oxidation of S35 - and CI4 -labeled thiouracil by chloroperoxidase . The reaction mixture contained 200 pmoles of phosphate buffer, pH 2 .75, 4 1~moles of hydro en peroxide, 0 .29 pg of chloroperoxidase plus 0 .8 moles of S3~-thiouracil (3 " 25 x 105 cPq~~lemole) in Experiment 1 and 0 .5 moles of CI4-thiouracil (2 x 10° cpm~~emole) in Experiment 2 . The total volume was 2 ml . The reaction mixtures were incubated at room temperature for 3 min, then frozen and lyopholized . The residue remaining after lyopholization was extracted with 0 .5 ml absolute alcohol and the alcohol extract spotted on Whatman No . 1 paper for chromatography . The chromatograms were developed in n-butanol-ammonia solvent (n-butanol saturated with 1 .5 N NH40H) . Thiouracil and uracil spots were detected by U .V . absorption . Radioactivity was determined on a Vanguard strip counter. for the oxidation of thiouracil, it is not incorporated into the fïnal oxidation products as evidenced by the lack of radioactivity in the products when KC1~ is used in the incubation medium .
In addition, under proper incubation conditions,
the halogen anion may function in catalytic quantities . Summary Chloroperoxide catalyzes the desulfurization of thiouracil when supplemented with hydrogen peroxide and a halogen anion .
Uracil and elemental sulfur are the
products of thiouracil oxidation . Thiouracil inhibits enzymatic halogenation reactions catalyzed by chloroperoxidase by competing with the substrate halogen-acceptor molecule for the
No .7
MECHANISM OF INHIBITION OF ENZYMATIC HALOGÉNATION
oxidized halogen ion-enzyme complex .
325
Extrapolation of these results to mammalian
systems suggests that thiouracil and related compounds inhibit the formation of thyroid hormone by competing with the tyrosine residues of thyroglobulin for enzyme-bound iodinium ion . Acknowledgment - This work has been supported by a grant (No . G-18741) from the National Science Foundation . References 1.
? 2560 (1959) " P .D . SHAW, J.R . BECKWITH and L .P . HAGER, J. Biol . Chem . 34,
2.
P .D . SHAW and L.P . HAGER, J. Biol . Chi. X35,, 2565 (1959) "
3"
$.D . SHAW and L.P . HAGER, J. Amer . Chew . Soc. ~1, 6527 (1959) "
l, .
P.D . SHAW and L .P . HAGER, J . Biol . Chem . ? 3¢, 1626 (1960) .
5.
H. EBERWEIN and L.P . HAGER, Abstr. of Amer . Chem . Soc . Keeting . St . Louis . o.
6.
p . 7c (1961) .
J .W . REINER, Behavior of Enzyme Systems p . 170 .
Burgess, Minneapolis (1959) "
Pergamon Press Ltd.
Printed in
ON THE MECHANISM OF INHIBITION OF ENZYMATIC HALOGÉNATION BY ANTITHYROID AGENTS David Morris, Horst Eberwein and Lowell P . Hager Biochemistry Division, University of Illinois, Urbana, Illinois (Received 20 June 1962) CHLOROPEROXIDASE preparations oxidize chloride, l-4 iodide and bromide ion with the formation of the corresponding carbon-halogen bond when supplied with a suitable acceptor molecule . 5
This finding draws a close analogy to the formation
of thyroid hormone which is further strengthened by the observation that chloroperoxidase action is inhibited by the classical antithyroid agents such as thiourea and thiouracil .2
The mechanism by which these compounds inhibit enzy-
matic halogenation is not known although several hypotheses have been suggested on the basis of experimental findings arising from studies concerning the biosynthesis of thyroid hormone . The chloroperoxidase system offers several advantages for a study of. the mechanism of inhibition of enzymatic halogenation by these antithyroid agents especially with 1,1-dimethyl-4-chloro-3,5-cyclohexanedione (monochlorodimedone), chloride ion and hydrogen peroxide as substrates (equation 1) .
Firstly, the
product of the enzymatic halogenation reaction, 1,1-dimethyl-4,4-dichloro-3,5-~ cyclohexanedione (dichlorodimedone), can be readily measured by either an optical or radioactive assay .
Secondly, with either chloride or bromide ion as substrate
the kinetics of the halogen reaction are not complicated by the non-enzymatic oxidation of the halogen anion such as occurs with iodide and hydrogen peroxide . ÇI
CI + C1
_
+ Hy02 + H+ ---~-
321
CI
0 0 ~ + ZHZO
(1)
èIECHANISM OF INHIBITI(~i OF ENZYMATIC HALOGÉNATION
322
No .7
A series of experimental findings with the chloroperoxidase reaction prompts us to propose a general mechanism for the inhibition of enzymatic halogenation by thiouracil . z
80
0 W O W W z O C W O O _U
0
500 I
1000
/ MONOCHLORODIMEDONE
1500 (MOLES / LITER)
FIG . 1 Effect of thiouracil concentration on the enzymatic formation of dichlorodimedone at various concentrations of monochlorodimedone . The complete system (~~) contained the components listed in Table 1 at the indicated concentrations of monochlorodimedone . The effect of increasing amounts of thiouracil on the enzymatic chlorination reaction is shown in curves 2, 3 and 4, where thio uracil is present in the following concentrationss ( - ~ 1 x lo'~ M ; (~-~) z x l0'4 M ; (~-~) 4 x l0'4 M .
Q Q
Resu is Table 1 shows the inhibition of dichlorodimedone formation by thiouracil and the reversal of this inhibition by increasing amounts of monochlorodimedone . Figure 1 graphically presents a series of experimental curves resulting from a plot of the reciprocal of the rate of dichlorodimedone formation against the reciprocal of the monochlorodimedone concentration at various levels of
No .7
MECHANISM OF INHIBITION OF ENZYMATIC HALOGÉNATION
323
TABLE 1 Inhibition of Dichlorodimedone Formation by Thiouracil and its Reversal by Monochlorodimedone Monochlorodimedone added
Dichlorodimedone formation
(pmoles)
1.
0 .5
179
2.
2 .5
202
3.
5 .0
214
4"
10 .0
5.
20 .0
194
6.
40 .0
190 194
The complete system conta}qed 500 pmoles of potassium phosphate buffer, pH 2 .75, 50 moles of KC1 (22,400 cpm/mole), 10 umoles of hydrogen peroxide, 0.5 ~9 chloroperoxidase plus the indicated amounts of monochlorodimedone in a total volume of 5 ml . In reactigns containing thioùracil, it was present at a concentration of 2 x 10 - M. The reaction mixtures were incubated at 30 ° for 4 min. Optical assays indicate that the rate of dichlorodimedone formation is constant during this 4 min incubation period . The reaction was stopped by the addition of 1 ml of 7 N sulfuric acid . Dichlorodimedone formation was measured by exhaustive extraction of the reaction mixtures with ether and determination of the radioactivity in the ether phase . Dichlorodimedone was shown to be the product of the reaction by repeated crystallization of the radioactive enzymatic product with synthetic dichlorodimedone . When 106,500 cpm of radioactive product was mixed with 200 mg of synthetic non-radioactive dichlorodimedone, the specific activity (cpm/mg) after successive crystallizations from petroleum ether was 534, 529, 517, 524 . thiouracil .
These curves are typical of those cases where competitive inhibition
is brought about by a competing substrate . 6 The major end-product of this reaction has been identified as uracil by cochromatography with authentic uracil and by a comparison of the radioactive products obtained from C U`- and S35 -labeled thiouracil (Fig . 2) .
Paper chromato
grams show that at least 80 per cent of the C 14-thiouracil is converted to C 14 uracil .
In contrast, when S35 -thiouracil is used as substrate, no radioactivity
is associated with the uracil spot on the paper chromatogram .
Present evidence
indicates that a majority of the sulfur from thiouracil is converted to elemental sulfur . Although a halogen anion (i .e ., chloride, bromide or iodide) is required
324
MECHANISM OF INHIBITION OF ENZYMATIC HALOGÉNATION
~ 0
1
EXPERIMENT
I
S"= THIOURACIL
ô
10 _EXPERIMENT 2
C I4 -THIOURACIL
No .7
MARKERS I
0 o~lalN
I
THIOURACIL
QI
I
0.2
I
0.3
URACIL I
I
I
0.4
O.s
0.8
Rf VALUES
1
L0 s v r
FIG . 2 Oxidation of S35 - and CI4 -labeled thiouracil by chloroperoxidase . The reaction mixture contained 200 pmoles of phosphate buffer, pH 2 .75, 4 1~moles of hydro en peroxide, 0 .29 pg of chloroperoxidase plus 0 .8 moles of S3~-thiouracil (3 " 25 x 105 cPq~~lemole) in Experiment 1 and 0 .5 moles of CI4-thiouracil (2 x 10° cpm~~emole) in Experiment 2 . The total volume was 2 ml . The reaction mixtures were incubated at room temperature for 3 min, then frozen and lyopholized . The residue remaining after lyopholization was extracted with 0 .5 ml absolute alcohol and the alcohol extract spotted on Whatman No . 1 paper for chromatography . The chromatograms were developed in n-butanol-ammonia solvent (n-butanol saturated with 1 .5 N NH40H) . Thiouracil and uracil spots were detected by U .V . absorption . Radioactivity was determined on a Vanguard strip counter. for the oxidation of thiouracil, it is not incorporated into the fïnal oxidation products as evidenced by the lack of radioactivity in the products when KC1~ is used in the incubation medium .
In addition, under proper incubation conditions,
the halogen anion may function in catalytic quantities . Summary Chloroperoxide catalyzes the desulfurization of thiouracil when supplemented with hydrogen peroxide and a halogen anion .
Uracil and elemental sulfur are the
products of thiouracil oxidation . Thiouracil inhibits enzymatic halogenation reactions catalyzed by chloroperoxidase by competing with the substrate halogen-acceptor molecule for the
No .7
MECHANISM OF INHIBITION OF ENZYMATIC HALOGÉNATION
oxidized halogen ion-enzyme complex .
325
Extrapolation of these results to mammalian
systems suggests that thiouracil and related compounds inhibit the formation of thyroid hormone by competing with the tyrosine residues of thyroglobulin for enzyme-bound iodinium ion . Acknowledgment - This work has been supported by a grant (No . G-18741) from the National Science Foundation . References 1.
? 2560 (1959) " P .D . SHAW, J.R . BECKWITH and L .P . HAGER, J. Biol . Chem . 34,
2.
P .D . SHAW and L.P . HAGER, J. Biol . Chi. X35,, 2565 (1959) "
3"
$.D . SHAW and L.P . HAGER, J. Amer . Chew . Soc. ~1, 6527 (1959) "
l, .
P.D . SHAW and L .P . HAGER, J . Biol . Chem . ? 3¢, 1626 (1960) .
5.
H. EBERWEIN and L.P . HAGER, Abstr. of Amer . Chem . Soc . Keeting . St . Louis . o.
6.
p . 7c (1961) .
J .W . REINER, Behavior of Enzyme Systems p . 170 .
Burgess, Minneapolis (1959) "