- Email: [email protected]
Deiodination of thyroid hormone and the role of glutathione
TIBS -August 1980
222
it is labile to fieat and shows saturation kinetics. More recently, other reactions such as the conversion of T4 into rTa and of T3 or rT3 into 3,3'-'I"2, have also been investigated in vitro 4'5. In the study of the subcellular location of'l"4 5'-deiodinase Theo J. Visser activity in rat liver homogenates, it was noted that the enzyme in the microsomal The enzymatic conversion o f thyroxine into the more potent biological compound, 3,3',5- fraction required a soluble factor for activtri-iodothyronine (Ts), takes place in many tissues and requires thiols. Reduced glutathione ity. This factor could only be replaced by (GSH) may be the endogenous cofactor. Recent evidence suggests that the decreased thiols, such as dithiothreitol (DTI') and production o f Ts which can occur, for example, during starvation, is caused-at least in part ethanethiol suggesting that deiodination of "1"4is a reductive process: T4 + 2 RSH --*T3 - by reduced concentrations ofintracellular GSH. + RS-SR + HI e. Reduced glutathione Thyroxine (T4) is the main secretory prod- process because the activity of the 5'- (GSH), though less effective, also supports uct of the thyroid and though a small deiodinase can be reduced selectively in the conversion ofT4 into "1"3and it may well amount of 3,3',5-tri-iodothyronine (I'3) is several situations, notably during dietary be the endogenous cofactor for this reacalso secreted, most of the circulating 3"3 is restriction 2. This causes a decrease in the tion. Deiodinase activity is inhibited by derived from deiodination of the phenolic concentration of serum T3 but, in general, agents which block sulfhydryl groups 4,6and inhibition is not relieved by subsequent ring (5'-deiodination) of '1"4 in peripheral an increase of rT3. addition of an excess of DTT 6. The enzyme tissues. Alternatively, 5-deiodination of is stimulated by EDTA 5,e. These findings the tyrosyl ring of "1"4yields 3,3 ',5 '-tri- Characteristic features of deiodinafing strongly suggest that cysteine residues are iodothyronine (reverse '1"3, rT3), a sub- enzymes Since radioimmunoassay has been used essential for enzyme activity.. stance not secreted by the thyroid in subReverse T3 (and also 3',5'-T2, Ref. 1) is a stantial quantities. Both Ts and rT, are to study the conversion of T4 into "1"3by r a t deiodinated further to 3,5-, 3,3'- and liver homogenates', the subject has competitive inhibitor of the 5'-deiodin3',5'-di-iodothyronine (T2), then to 3- and aroused the interest of many investigators. ation of T4 and Vice versa, where values for 3'-iodothyronine (Tx) and finally to It was soon established that this reaction, K= and Ki have been shown to be identhyronine (To) (Fig. 1) 1. Of all these com- which may also be observed in other tissue tical ~'8.In contrast, 3,5-'1"2hardly affects the pounds Ts is by far the most biologically preparations (notably kidney homogen- conversion of "1"4into T3x. These results are with the two-enzyme active. It is difficult to estimate precisely ate), is catalysed by enzymes since compatible the relative activities of "1"4and "1"3because the former is converted into the latter. However, comparison of their biological potencies in vivo, indicates that T~ is 3-5 c.2_c._coo. times as active as '1"4. Since about 30% of the turnover of T4 is directed towards the ;3' 2' ;3 2 production of "1"3,this indicates that "1"4has little if any intrinsic activity. Reverse T~ is I I I I virtually devoid of thyromimetic activity. Studies in vitro (see below) have shown that rTs is eliminated mainly by 5'deiodination to 3,3'-T=. Production of T3 T31 and degradation of rT3 are, therefore, similar reactions, probably mediated by a single I I I I enzyme (iodothyronine 5'-deiodinase). This enzyme would need to be distinguished from iodothyronine 5-deiodinase, which would catalyse, for example, the I 3',5'-T2 3,3'-T2 3,542 I production of rTs from T4 and of 3,3'-'1"2 from "1"3. The entire sequential deiodini t ation of T, could result from the action of just these two enzymesk Oxidative deamination and conjugation to sulfates and glucuronides also contribute to the 3'~ 1 341 metabolism of iodothyronines, albeit to a lesser extentk ,it- - ~ j Deiodination is not an uncontrolled
Deiodination of thyroid hormone and the role of glutathione
T. J. Visser is at the Department of lnternal Medicine III "and Clinical Endocrinology, Medical Faculty, Erasmus University, P.O. Box 1738, Rotterdam, The Netherlands.
TO Fig. 1. Metabolism o f thyroxine (3,3 "5,5'-tetraiodothyronine, 1"4)by sequential 5-( / ) and 5'-deiodination ( x, ). 9 Elsevier/North-Holland Biomedical Press 1980
223
TIBS -August 1980
hypothesis. Of the many reactions, 5'deiodination of rT3 appears to be catalysed most effectively. Compared with T4 as substrate, K= values are considerably lower 9and V ~ values are substantially higher (0.06 v. 2.3 p.M and 560 v. 30 pmol min -~ per mg of microsomal protein, respectively; 37~ pH 7.2, 3 mM DTT) 8. Thus, while formation rates are comparable, rT3 is degraded more rapidly than T3, the most biologically effective form. Pronounced effects of pH on the deiodinase activity hav~ been observed. In general, 5-deiodination is optimal at about pH 8, whereas the influence of pH on 5'deiodinase activity is dependent on the substrate and its concentration, but at low (physiological) concentrations the reaction rate is invariably maximal at about pH 6.56. The exact subcellular location of the enzymes has been investigated with apparently conflicting results. In rat kidney, T, 5'-deiodinase activity was found to be associated with the plasma membrane fraction ~. This finding was confirmed by one group of investigators t~ but contradicted by two others ~,t2 for the enzyme from rat liver. Here the deiodinase activity was shown to be located within the endoplasmic reticulum. The physiological relevance of studies in vitro is emphasized by the potent inhibitory effect of derivatives of 2-thiouracil (TU) ',5.7 which have also been found to inhibit 5'deiodinase activity in vivo 7. T h e structure-activity relationships of these and related compounds as determined from experiments in vivo and in vitro are very similar Is,". These thioureylenes (having in common the = N - C(:S) - N = structure) are also potent inhibitors of the biosynthesis of thyroid hormone because they lower the activity of thyroid peroxidase. This enzyme catalyses the iodination of tyrosine residues in thyroglobulin and the subsequent coupling of iodotyrosines to iodothyronines. Among this group of inhibitors are 6-propyl-2-thiouracil (PTU) and 2-mercapto-l-methylimidazole (thiamazole), which are used in the treatment of patients with hyperthyroidism. In contrast to PTU, however, thiamazole hardly 9 affects deiodination of thyroid hormone either bt vivo ~3 or in vitro ~. Mechanism of deiodination of thyroid
hormone Recent observations (summarized in Fig. 2) have given an insight into the mechanism of action of iodothyronine 5'-deiodinase. TU is a dead-end inhibitor of the 5'-deiodination of both "1"4and rT3 and this inhibition is uncompetitive with
respect to substrate '.15.16 but competitive with respect to cofactor (DTT)".". A change in the cofactor concentration results in a parallel displacement of the Lineweaver-Burk plot of the deiodination rate as a function of the substrate concentration 15'a6. Deiodination would therefore seem'to follow a ping-pong mechanism, involving the formation of an intermediate enzyme complex which is acted upon by the cofactor with the regeneration of free enzyme, or which may form a dead-end complex with TU. The chemical nature of the cofactor and of the inhibitor with its strict requirement for the 2-mercapto group", and the presence of essential cysteine residues in the enzyme point to the possible oxidation of enzyme-sulfhydryl groups during deiodination. This is sup-
++
(A)
I/V ~
+
PTU
Vs
(B)
++
ported by the work of Cunninghan iT, who has shown that TU reacts selectively with protein-sulfenyl iodide (-SI) groups leading to the formation of protein-thiouracil mixed disulfides. The reaction pathway may, therefore, be visualized as consisting of two half-reactions: "I"4+ E-SH ~'I"3 + E-SI E-SI + 2 R-SH ~ E-SH + R-S-S-R + HI (where E-SH represents free enzyme). It should be noted that the reduction of the E-SI intermediate requires two thiol groups of the cofactor. These are supplied by one molecule of DTT. Inhibition by thiouracil (X-SH) would result from the following reaction: E-SI + X-SH ~ E~
+ HI
A prediction of this model is that thiouracils would only react with the enzyme in the presence of substrate. This is true since binding of radioactive PTU to rat liver microsomes was specifically induced by iodothyronines, of which the 'better' substrates, rT3 and 3',5'-T~, were the most active TM. Moreover, irreversible inactivation of the enzyme by thiouracils required the presence of substrate 15. Since 5-deiodination is also stimulated by thiols and'inhibited by TU 5, the above pathway may apply to the deiodination of thyroid hormone in general.
1/v
PTU
1/cofactor (c)
+
1/v
++
cofactor
+++
1/s Fig. 2. Kinetics of enzymatic 5'-deiodination and the effects o f derivatives of 2-thiouracil (e.g. 6propyl-2-thiouracil, 6-PTU). (A) Inhibition by PTU is uncompetitive with respect to substrate. (B) Inhibition by PTU is competitive with respect to cofactor (e.g. DTT). (C) K,n and Vmaxincrease proportionally with an increase in cofactor concentration.
Physiological hnplications The deiodinase activity of tissue homogenates reflects the state of thyroid hormone metabolism in the donor and can be influenced by several treatments. Specifically, liver homogenates from fasted rats have a decreased activity of 5'deiodinase. Differences between fed and fasted rats, however, were not detected when 5'-deiodinase activity was assayed in the presence of DqT 19. Rat liver cytosol from fasted rats supported conversion of "1"4 into T3 by microsomes less effectively than cytosol from fed rats 2~ The defect in.the cytosol from fasted rats could be restored by the addition of GSH or NADPH 2~ A decrease in the concentration of GSH in 'fasted' homogenates could also be measured ag. These findings support the role of GSH as cofactor in the deiodination of iodothyronines. The lower levels of GSH during fasting may be caused by an impaired functioning of the hexose monophosphate shunt, which normally synthesizes NADPH. Since NADPH is the cofactor in the reduction of oxidized glutathione (GSSG) to GSH by glutathione reductase, intracellular GSH concentrations
224 and therefore 5'-deiodinase activity are lowered during fasting. Depletion of cysteine - the essential amino acid in glutathione - may also play a contributory role 2x. Because GSSG inhibits deiodination I it is not clear at the moment whether the concentration of GSH per se or the ratio [GSIt]/[GSSG] is the most important factor in the regulation of the deiodination process. The decrease in the production of T3 may be regarded as beneficial during fasting since it results in diminished energy expenditure and tissue breakdown when the body is in a catabolic state. Though thiols stimulate and thiouracils inhibit both 5- and 5'-deiodinations, fasting and PTU administration lead to a selective decrease in the activity of 5'deiodinase and therefore a decrease in serum "1"3and and increase in serum rT~. It may of course be argued that reactions catalysed by 5'-deiodinase are more sensitive to conditions which intervene in the enzymatic cycle at the site of E-SI reduction by cofactor. In the absence of direct experimental evidence, however, this must remain purely speculative. Additional regulatory mechanisms have been suggested such as changes in substrate availability by effects on tissue uptake ~2 and intracellular binding of iodothyronines ~. Changes in intracellular pH may also be of importance ~. Nevertheless, the role of GSH in thyroid hormone metabolism appears to be established. The evidence that two enzymes are involved (iodothyronine 5and 5'-deiodinases) in the sequential deiodination of T4 is, however, circumstantial; a proof depends on their eventual purification.
TIBS -August 1980 10 Madel, R. M. B., Ozawa, Y. and Chopra, I. J. (1979) Endocrinology 104, 365-371 11 Auf dem Brinke, D., Hesch, R-D. and K6hrle, J. (1979) Biochem. 1. 180, 273-279 12 Fekkes, D., Van Overmeeren-Kaptein, E., Docter, R., Hennemann, G. and Visser, T. J. (1979) Biochim. Biophys. Acts 587, 12-19 13 Hershman, J. M. and Van Middlesworth, L. (1962) Endocrfnology 71, 94-I O0 14 Visser, T. J., Van Overmeeren, E., Fekkes, D., Docter, R. and ltennemann, G. (1979) FEBS Len. 103, 314--318 15 Leonard, J. L. and Rosenberg, I. N. (1978) Endocrinology 103, 2137-2144 16 Visser, T. J. (1979) Biochirn. Biophys. Acts 569,
302-308
Structure and cooperativity of haemoglobin Joyce Baldwin Tertiary and quaternary structural changes hz haemoglobin are coupled in a way that suggests an underlying two.state allosteric mechanism for its cooperative ligand bindhtg.
Since Perutz' first proposed a stereochemical mechanism for cooperative oxygen binding to haemoglobin, certain relationships between the atomic structure of haemoglobin and its function have become well established. Comparison of the structures o'f deoxy- and fully liganded haemoglobin as determined by X-ray crystallography suggests that the major part of the cooperativity of haemoglobin is achieved by the molecule changing its quaternary structure when ligands bind, as envisaged in the two-state aUosteric mechanism References proposed by Monod, Wyman and I Chopra, I. J., Solomon, D. H., Chopra, U., Wu, Changeux 2. In such a mechanism, two S. Y., F'tsher, D.A. and Nakamura, Y. (1978) alternative quaternary structures having Rec. Prog. ttorm. Res. 34, 521-567 2 Suda, A.K., Pinman, C.S., Shimizu, T. and different affinities for ligand are in equilibrium with each other at all stages of ligaChambers, J. B. (1978)J. Clin. EndocrinoL Metab. 47, 1311-1319 tion, and the binding of the ligand swings 3 Hesch, R-D., Brunner, G. and Soling, H.D. the equilibrium towards the high affinity (1975) Clin. Chim. Acta 59, 209-213 form. It is known that, in haemoglobin, the 4 Chopra, I. J., Wu., S.Y., Nakamura, Y. and Solomon, D.H. (1978) Endocrinology 102, affinity of each of the 'two states' is altered slightly by changes in conditions, such as 1099-1105 5 Visser, T.J., Fekkes, D., Decter, R. and change in pH or salt concentration, but Hennemann, G. (1978) Biochem. J. 174, these alterations arise from small modifica221-229 tions to each of two essentially distinct 6 Visser, T. J., Van tier Does-Tobr, I., Docter, R. structures. It is not yet clear whether there and Hennemann, G. (1976) Biochem. Z 157, are significant cooperative interactions in 479-482 7 Kaplan, M. M. and Utiger, R. D. (1978)1. Clin. haemoglobin that are not coupled to the Invest. 61,459--471 quaternary change. 8 Visser, T. J., Fekkes, D., Docter, R. and The change in quaternary structure, or Hennemann, G. (1979) Biochem. J. 179, 489--495 9 Leonard, J.L. and Rosenberg, I.N. (1978) Endocrinology 103,274-280
17 Cunningham, L. W. (1964) Biochemistry 3, 1629-1634 18 Visser, T. J. and Van Overmeeren, E. (1979) Biochem. 1. 183, 167-169 19 Harris, A. R. C., Fang, S. L., Hinerfeld, L., Braverman, L. E. and Vagenakis, A. G. (1979) J. Clin. Invest. 63,516-524 20 Balsam, A. and lngbar, S. H. (1979) J. Clin. Invest. 63, 1145-1156 21 Higashi, T., Tateishi, N., Naruse, A. and Samamoto, Y. (1977)1. Biochem. 82, 117-124 22 Jennings, A. S., Ferguson, D. C. and Utiger, R. D. (1979)1. Clin. Invest. 64, 1614-1623 23 Hrftken, B., Krdding, R. and Hesch, R-D. (I 977) Clin. Chirn. Acta 78,261-266
Joyce Baldwin is at the MRC Laboratory of Molecular Biology, Hills Rd, Cambridge, U.K.
rearrangement of the four subunits, was first observed in the low-resolution structures of haemoglobin ~. Deoxyhaemoglobin has a unique quaternary structure, whereas haemoglobins in which ligands, such as oxygen, carbon monoxide or water, are bound to the iron atoms of the haems aU have the same quaternary structure. Comparison of the structures of horse and human deoxyhaemoglobin with that of horse liganded haemoglobin when these structures were known to 2.8, 3.5 and 2.8 /~ resolution respectivelyH showed that the main features of the tertiary structures of the subunits were similar in deoxyand liganded haemoglobin, in spite of the quaternary change (see Fig. 1). The most striking differences between the structures of deoxy- and liganded haemoglobin that were first seen to be important were the difference in position of each iron atom relative to the mean plane of its haem group, and the presence of inter-dimer salt-bridges in deoxy-, but not in liganded, haemoglobin. The nature of the linkage between quaternary structure and the position of the iron atoms was not clear, buta mechanism was proposed in the framework of a two-state allosteric model, in which the additional bonds that stabilize the low-affinity form were identified with the salt-bridges t. The structures of horse met- and human deoxyhaemoglobin are now known at higher resolution, 2.0 and 9 Elsevier/North-HollandBiomedicalPress1980
222
it is labile to fieat and shows saturation kinetics. More recently, other reactions such as the conversion of T4 into rTa and of T3 or rT3 into 3,3'-'I"2, have also been investigated in vitro 4'5. In the study of the subcellular location of'l"4 5'-deiodinase Theo J. Visser activity in rat liver homogenates, it was noted that the enzyme in the microsomal The enzymatic conversion o f thyroxine into the more potent biological compound, 3,3',5- fraction required a soluble factor for activtri-iodothyronine (Ts), takes place in many tissues and requires thiols. Reduced glutathione ity. This factor could only be replaced by (GSH) may be the endogenous cofactor. Recent evidence suggests that the decreased thiols, such as dithiothreitol (DTI') and production o f Ts which can occur, for example, during starvation, is caused-at least in part ethanethiol suggesting that deiodination of "1"4is a reductive process: T4 + 2 RSH --*T3 - by reduced concentrations ofintracellular GSH. + RS-SR + HI e. Reduced glutathione Thyroxine (T4) is the main secretory prod- process because the activity of the 5'- (GSH), though less effective, also supports uct of the thyroid and though a small deiodinase can be reduced selectively in the conversion ofT4 into "1"3and it may well amount of 3,3',5-tri-iodothyronine (I'3) is several situations, notably during dietary be the endogenous cofactor for this reacalso secreted, most of the circulating 3"3 is restriction 2. This causes a decrease in the tion. Deiodinase activity is inhibited by derived from deiodination of the phenolic concentration of serum T3 but, in general, agents which block sulfhydryl groups 4,6and inhibition is not relieved by subsequent ring (5'-deiodination) of '1"4 in peripheral an increase of rT3. addition of an excess of DTT 6. The enzyme tissues. Alternatively, 5-deiodination of is stimulated by EDTA 5,e. These findings the tyrosyl ring of "1"4yields 3,3 ',5 '-tri- Characteristic features of deiodinafing strongly suggest that cysteine residues are iodothyronine (reverse '1"3, rT3), a sub- enzymes Since radioimmunoassay has been used essential for enzyme activity.. stance not secreted by the thyroid in subReverse T3 (and also 3',5'-T2, Ref. 1) is a stantial quantities. Both Ts and rT, are to study the conversion of T4 into "1"3by r a t deiodinated further to 3,5-, 3,3'- and liver homogenates', the subject has competitive inhibitor of the 5'-deiodin3',5'-di-iodothyronine (T2), then to 3- and aroused the interest of many investigators. ation of T4 and Vice versa, where values for 3'-iodothyronine (Tx) and finally to It was soon established that this reaction, K= and Ki have been shown to be identhyronine (To) (Fig. 1) 1. Of all these com- which may also be observed in other tissue tical ~'8.In contrast, 3,5-'1"2hardly affects the pounds Ts is by far the most biologically preparations (notably kidney homogen- conversion of "1"4into T3x. These results are with the two-enzyme active. It is difficult to estimate precisely ate), is catalysed by enzymes since compatible the relative activities of "1"4and "1"3because the former is converted into the latter. However, comparison of their biological potencies in vivo, indicates that T~ is 3-5 c.2_c._coo. times as active as '1"4. Since about 30% of the turnover of T4 is directed towards the ;3' 2' ;3 2 production of "1"3,this indicates that "1"4has little if any intrinsic activity. Reverse T~ is I I I I virtually devoid of thyromimetic activity. Studies in vitro (see below) have shown that rTs is eliminated mainly by 5'deiodination to 3,3'-T=. Production of T3 T31 and degradation of rT3 are, therefore, similar reactions, probably mediated by a single I I I I enzyme (iodothyronine 5'-deiodinase). This enzyme would need to be distinguished from iodothyronine 5-deiodinase, which would catalyse, for example, the I 3',5'-T2 3,3'-T2 3,542 I production of rTs from T4 and of 3,3'-'1"2 from "1"3. The entire sequential deiodini t ation of T, could result from the action of just these two enzymesk Oxidative deamination and conjugation to sulfates and glucuronides also contribute to the 3'~ 1 341 metabolism of iodothyronines, albeit to a lesser extentk ,it- - ~ j Deiodination is not an uncontrolled
Deiodination of thyroid hormone and the role of glutathione
T. J. Visser is at the Department of lnternal Medicine III "and Clinical Endocrinology, Medical Faculty, Erasmus University, P.O. Box 1738, Rotterdam, The Netherlands.
TO Fig. 1. Metabolism o f thyroxine (3,3 "5,5'-tetraiodothyronine, 1"4)by sequential 5-( / ) and 5'-deiodination ( x, ). 9 Elsevier/North-Holland Biomedical Press 1980
223
TIBS -August 1980
hypothesis. Of the many reactions, 5'deiodination of rT3 appears to be catalysed most effectively. Compared with T4 as substrate, K= values are considerably lower 9and V ~ values are substantially higher (0.06 v. 2.3 p.M and 560 v. 30 pmol min -~ per mg of microsomal protein, respectively; 37~ pH 7.2, 3 mM DTT) 8. Thus, while formation rates are comparable, rT3 is degraded more rapidly than T3, the most biologically effective form. Pronounced effects of pH on the deiodinase activity hav~ been observed. In general, 5-deiodination is optimal at about pH 8, whereas the influence of pH on 5'deiodinase activity is dependent on the substrate and its concentration, but at low (physiological) concentrations the reaction rate is invariably maximal at about pH 6.56. The exact subcellular location of the enzymes has been investigated with apparently conflicting results. In rat kidney, T, 5'-deiodinase activity was found to be associated with the plasma membrane fraction ~. This finding was confirmed by one group of investigators t~ but contradicted by two others ~,t2 for the enzyme from rat liver. Here the deiodinase activity was shown to be located within the endoplasmic reticulum. The physiological relevance of studies in vitro is emphasized by the potent inhibitory effect of derivatives of 2-thiouracil (TU) ',5.7 which have also been found to inhibit 5'deiodinase activity in vivo 7. T h e structure-activity relationships of these and related compounds as determined from experiments in vivo and in vitro are very similar Is,". These thioureylenes (having in common the = N - C(:S) - N = structure) are also potent inhibitors of the biosynthesis of thyroid hormone because they lower the activity of thyroid peroxidase. This enzyme catalyses the iodination of tyrosine residues in thyroglobulin and the subsequent coupling of iodotyrosines to iodothyronines. Among this group of inhibitors are 6-propyl-2-thiouracil (PTU) and 2-mercapto-l-methylimidazole (thiamazole), which are used in the treatment of patients with hyperthyroidism. In contrast to PTU, however, thiamazole hardly 9 affects deiodination of thyroid hormone either bt vivo ~3 or in vitro ~. Mechanism of deiodination of thyroid
hormone Recent observations (summarized in Fig. 2) have given an insight into the mechanism of action of iodothyronine 5'-deiodinase. TU is a dead-end inhibitor of the 5'-deiodination of both "1"4and rT3 and this inhibition is uncompetitive with
respect to substrate '.15.16 but competitive with respect to cofactor (DTT)".". A change in the cofactor concentration results in a parallel displacement of the Lineweaver-Burk plot of the deiodination rate as a function of the substrate concentration 15'a6. Deiodination would therefore seem'to follow a ping-pong mechanism, involving the formation of an intermediate enzyme complex which is acted upon by the cofactor with the regeneration of free enzyme, or which may form a dead-end complex with TU. The chemical nature of the cofactor and of the inhibitor with its strict requirement for the 2-mercapto group", and the presence of essential cysteine residues in the enzyme point to the possible oxidation of enzyme-sulfhydryl groups during deiodination. This is sup-
++
(A)
I/V ~
+
PTU
Vs
(B)
++
ported by the work of Cunninghan iT, who has shown that TU reacts selectively with protein-sulfenyl iodide (-SI) groups leading to the formation of protein-thiouracil mixed disulfides. The reaction pathway may, therefore, be visualized as consisting of two half-reactions: "I"4+ E-SH ~'I"3 + E-SI E-SI + 2 R-SH ~ E-SH + R-S-S-R + HI (where E-SH represents free enzyme). It should be noted that the reduction of the E-SI intermediate requires two thiol groups of the cofactor. These are supplied by one molecule of DTT. Inhibition by thiouracil (X-SH) would result from the following reaction: E-SI + X-SH ~ E~
+ HI
A prediction of this model is that thiouracils would only react with the enzyme in the presence of substrate. This is true since binding of radioactive PTU to rat liver microsomes was specifically induced by iodothyronines, of which the 'better' substrates, rT3 and 3',5'-T~, were the most active TM. Moreover, irreversible inactivation of the enzyme by thiouracils required the presence of substrate 15. Since 5-deiodination is also stimulated by thiols and'inhibited by TU 5, the above pathway may apply to the deiodination of thyroid hormone in general.
1/v
PTU
1/cofactor (c)
+
1/v
++
cofactor
+++
1/s Fig. 2. Kinetics of enzymatic 5'-deiodination and the effects o f derivatives of 2-thiouracil (e.g. 6propyl-2-thiouracil, 6-PTU). (A) Inhibition by PTU is uncompetitive with respect to substrate. (B) Inhibition by PTU is competitive with respect to cofactor (e.g. DTT). (C) K,n and Vmaxincrease proportionally with an increase in cofactor concentration.
Physiological hnplications The deiodinase activity of tissue homogenates reflects the state of thyroid hormone metabolism in the donor and can be influenced by several treatments. Specifically, liver homogenates from fasted rats have a decreased activity of 5'deiodinase. Differences between fed and fasted rats, however, were not detected when 5'-deiodinase activity was assayed in the presence of DqT 19. Rat liver cytosol from fasted rats supported conversion of "1"4 into T3 by microsomes less effectively than cytosol from fed rats 2~ The defect in.the cytosol from fasted rats could be restored by the addition of GSH or NADPH 2~ A decrease in the concentration of GSH in 'fasted' homogenates could also be measured ag. These findings support the role of GSH as cofactor in the deiodination of iodothyronines. The lower levels of GSH during fasting may be caused by an impaired functioning of the hexose monophosphate shunt, which normally synthesizes NADPH. Since NADPH is the cofactor in the reduction of oxidized glutathione (GSSG) to GSH by glutathione reductase, intracellular GSH concentrations
224 and therefore 5'-deiodinase activity are lowered during fasting. Depletion of cysteine - the essential amino acid in glutathione - may also play a contributory role 2x. Because GSSG inhibits deiodination I it is not clear at the moment whether the concentration of GSH per se or the ratio [GSIt]/[GSSG] is the most important factor in the regulation of the deiodination process. The decrease in the production of T3 may be regarded as beneficial during fasting since it results in diminished energy expenditure and tissue breakdown when the body is in a catabolic state. Though thiols stimulate and thiouracils inhibit both 5- and 5'-deiodinations, fasting and PTU administration lead to a selective decrease in the activity of 5'deiodinase and therefore a decrease in serum "1"3and and increase in serum rT~. It may of course be argued that reactions catalysed by 5'-deiodinase are more sensitive to conditions which intervene in the enzymatic cycle at the site of E-SI reduction by cofactor. In the absence of direct experimental evidence, however, this must remain purely speculative. Additional regulatory mechanisms have been suggested such as changes in substrate availability by effects on tissue uptake ~2 and intracellular binding of iodothyronines ~. Changes in intracellular pH may also be of importance ~. Nevertheless, the role of GSH in thyroid hormone metabolism appears to be established. The evidence that two enzymes are involved (iodothyronine 5and 5'-deiodinases) in the sequential deiodination of T4 is, however, circumstantial; a proof depends on their eventual purification.
TIBS -August 1980 10 Madel, R. M. B., Ozawa, Y. and Chopra, I. J. (1979) Endocrinology 104, 365-371 11 Auf dem Brinke, D., Hesch, R-D. and K6hrle, J. (1979) Biochem. 1. 180, 273-279 12 Fekkes, D., Van Overmeeren-Kaptein, E., Docter, R., Hennemann, G. and Visser, T. J. (1979) Biochim. Biophys. Acts 587, 12-19 13 Hershman, J. M. and Van Middlesworth, L. (1962) Endocrfnology 71, 94-I O0 14 Visser, T. J., Van Overmeeren, E., Fekkes, D., Docter, R. and ltennemann, G. (1979) FEBS Len. 103, 314--318 15 Leonard, J. L. and Rosenberg, I. N. (1978) Endocrinology 103, 2137-2144 16 Visser, T. J. (1979) Biochirn. Biophys. Acts 569,
302-308
Structure and cooperativity of haemoglobin Joyce Baldwin Tertiary and quaternary structural changes hz haemoglobin are coupled in a way that suggests an underlying two.state allosteric mechanism for its cooperative ligand bindhtg.
Since Perutz' first proposed a stereochemical mechanism for cooperative oxygen binding to haemoglobin, certain relationships between the atomic structure of haemoglobin and its function have become well established. Comparison of the structures o'f deoxy- and fully liganded haemoglobin as determined by X-ray crystallography suggests that the major part of the cooperativity of haemoglobin is achieved by the molecule changing its quaternary structure when ligands bind, as envisaged in the two-state aUosteric mechanism References proposed by Monod, Wyman and I Chopra, I. J., Solomon, D. H., Chopra, U., Wu, Changeux 2. In such a mechanism, two S. Y., F'tsher, D.A. and Nakamura, Y. (1978) alternative quaternary structures having Rec. Prog. ttorm. Res. 34, 521-567 2 Suda, A.K., Pinman, C.S., Shimizu, T. and different affinities for ligand are in equilibrium with each other at all stages of ligaChambers, J. B. (1978)J. Clin. EndocrinoL Metab. 47, 1311-1319 tion, and the binding of the ligand swings 3 Hesch, R-D., Brunner, G. and Soling, H.D. the equilibrium towards the high affinity (1975) Clin. Chim. Acta 59, 209-213 form. It is known that, in haemoglobin, the 4 Chopra, I. J., Wu., S.Y., Nakamura, Y. and Solomon, D.H. (1978) Endocrinology 102, affinity of each of the 'two states' is altered slightly by changes in conditions, such as 1099-1105 5 Visser, T.J., Fekkes, D., Decter, R. and change in pH or salt concentration, but Hennemann, G. (1978) Biochem. J. 174, these alterations arise from small modifica221-229 tions to each of two essentially distinct 6 Visser, T. J., Van tier Does-Tobr, I., Docter, R. structures. It is not yet clear whether there and Hennemann, G. (1976) Biochem. Z 157, are significant cooperative interactions in 479-482 7 Kaplan, M. M. and Utiger, R. D. (1978)1. Clin. haemoglobin that are not coupled to the Invest. 61,459--471 quaternary change. 8 Visser, T. J., Fekkes, D., Docter, R. and The change in quaternary structure, or Hennemann, G. (1979) Biochem. J. 179, 489--495 9 Leonard, J.L. and Rosenberg, I.N. (1978) Endocrinology 103,274-280
17 Cunningham, L. W. (1964) Biochemistry 3, 1629-1634 18 Visser, T. J. and Van Overmeeren, E. (1979) Biochem. 1. 183, 167-169 19 Harris, A. R. C., Fang, S. L., Hinerfeld, L., Braverman, L. E. and Vagenakis, A. G. (1979) J. Clin. Invest. 63,516-524 20 Balsam, A. and lngbar, S. H. (1979) J. Clin. Invest. 63, 1145-1156 21 Higashi, T., Tateishi, N., Naruse, A. and Samamoto, Y. (1977)1. Biochem. 82, 117-124 22 Jennings, A. S., Ferguson, D. C. and Utiger, R. D. (1979)1. Clin. Invest. 64, 1614-1623 23 Hrftken, B., Krdding, R. and Hesch, R-D. (I 977) Clin. Chirn. Acta 78,261-266
Joyce Baldwin is at the MRC Laboratory of Molecular Biology, Hills Rd, Cambridge, U.K.
rearrangement of the four subunits, was first observed in the low-resolution structures of haemoglobin ~. Deoxyhaemoglobin has a unique quaternary structure, whereas haemoglobins in which ligands, such as oxygen, carbon monoxide or water, are bound to the iron atoms of the haems aU have the same quaternary structure. Comparison of the structures of horse and human deoxyhaemoglobin with that of horse liganded haemoglobin when these structures were known to 2.8, 3.5 and 2.8 /~ resolution respectivelyH showed that the main features of the tertiary structures of the subunits were similar in deoxyand liganded haemoglobin, in spite of the quaternary change (see Fig. 1). The most striking differences between the structures of deoxy- and liganded haemoglobin that were first seen to be important were the difference in position of each iron atom relative to the mean plane of its haem group, and the presence of inter-dimer salt-bridges in deoxy-, but not in liganded, haemoglobin. The nature of the linkage between quaternary structure and the position of the iron atoms was not clear, buta mechanism was proposed in the framework of a two-state allosteric model, in which the additional bonds that stabilize the low-affinity form were identified with the salt-bridges t. The structures of horse met- and human deoxyhaemoglobin are now known at higher resolution, 2.0 and 9 Elsevier/North-HollandBiomedicalPress1980