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Is the association of concerted homotropic cooperative interactions and local heterotropic effects a general basic feature of regulatory enzymes?
BIOCHIMIE, 1981, 68, 103-105.
Is the association of concerted homotropic cooperative interactions and local heterotropic effects a general basic feature of regulatory enzymes ? Guy HERVt~.
Laboratoire d'Enzymologie, C.N.R.S., 91190 Gi[-sur-Yvette.
This article is dedicated to the memory of our friend and skillful biochemist Francois Seydoux.
Frangois Seydoux and his collaborators showed that phosphofructokinase from Saccharomyces cerevisiae is made of the association of catalytic and regulatory subunits [1]. From kinetic and spectroscopic studies of the binding of substrates and effectors these authors proposed a model associating concerted homotropic interactions and local heterotropic effects [2]. The same conclusion was reached by Nissler et aI. [3]. Independently a similar model has been proposed to account for the regulatory properties of Escherichia coli aspartate transcarbamylase [4] which is also constituted of catalytic and regulatory subunits [5]. This similarity might be of more general significance. It has been established that yeast phosphofructokinase is an octamer of two different kinds of polypeptides [1, 6, 7, 8] which were characterized as catalytic and regulatory chains on the basis of their AMP and ATP binding properties [1]. The conformational changes induced in the enzyme by these nucleotides and by the substrate fructose-6-phosphate were investigated by initial rate measurements and UV absorption or fluorescence difference spectroscopy. Fructose-6-phosphate appears to bind cooperatively to the catalytic sites of the enzyme and it is shown that this process involves a concerted transition. The effects of the activator AMP and of the inhibitor ATP appear not to involve co-operative interactions and it is concluded that these effectors act through a <~local site by site >>mechanism [2]. The inter-dependence between the effects of these two nucleotides lead the authors to conclude that these metabolites are shifting a single equilibrium in a reverse way. These conclusions are illustrated in the accompanying scheme. Since the inhibitory effect of ATP cannot be completely reversed by AMP it is still possible that the two nuc!eotides provoke distinct local conformational changes ; this possibility is illustrated in the model by dotted arrows (M. Laurent, personal communication).
Escherichia
coli
aspartate
transcarbamylase,
first enzyme of the pyrimidine pathway is made of the association of two catalytic trimers and three regulatory dimers. Catalytic and regulatory chains are different polypeptides. The structural and regulatory properties of this enzyme which shows homotropic and heterotropic interactions have been extensively reviewed [4, 9, 10]. The homotropic co-operative interactions between its catalytic sites for aspartate binding are interpreted in terms of a concerted transition. The catalytic activity is feedback inhibited by CTP and stimulated by ATP. In order to explain the properties of 2T'hioU-ATCase, (a modified form of the enzyme in which the homotropic co-operative interactions between the catalytic sites are selectively abolished) [11-14], and some characteristics of the process of stimulation of the normal enzyme activity by ATP [4] a model has been proposed. This model (represented in the accompanying scheme) associates a concerted transition corresponding to the homotropic co-operative interactions between the catalytic sites with <, local site by site ~>conformational changes corresponding to the action of the effectors ATP and CTP [4]. This interpretation was recently confirmed by the results of a series of elegant experiments showing that the T and R states of the enzyme are still sensitive to the action of ATP and CTP after being <~frozen ,> by means of cross-linking reagents which abolish the concerted transitions [15]. The models proposed account also for the influence of the effectors on the co-operativity between the catalytic sites, since their primary effect on the affinity of the catalytic sites for the substrate will change the degree of saturation of these sites and thus have a secondary effect on the T ~ R equilibrium via the substrate. The striking similarity in behaviour between yeast phosphofructokinase and E. coli aspartate transcarbamylase sug-
104
G. Hervd.
gests that the combination of concerted homotropic interactions and local heterotropic effects might be a general basic feature of the regulatory enzymes made of catalytic and regulatory subunits. Even in the case of enzymes in which the catalytic and regulatory sites are borne by the same polypeptidic chain it has been reported that the use of cross-linking reagents which aboslishes the co-operative interactions does not lead to the disappearance of the heterotropic effects between catalytic and regulatory sites. This is the case of rabbit muscle phosphofructokinase [16] and glycogen phosphorylase [17]. This phenomenon might be of significance concerning the Evolution of proteins. It appears that in a general way the polypeptidic structures cataCONCERTED
made of the aggregation of several of these polypeptides. More integrated multifunctional proteins then appeared, in which the catalytic sites are carried by the same polypeptide chain. Most probably the regulatory properties of protein evolved in the same way from structures resulting from the association of catalytic and regulatory subunits, to proteins in which catalytic and regulatory sites are borne by the same polypeptiJe chain. It seems that the association of catalytic and regulatory chains results in the possibibility of a concerted transition, the effect of which can be modulated by local conformational changes induced by effectors. The more integrated structures, in which catalytic and regulatory sites are formed by the same polypeptide chain, probably allows more complex interactions to occur, including homotropic posiTRANSITIONS fructose S.P
Aspartate I--,
n-
4.7
I
CTP
I.I.I I--
Aspartat~
::ATP
ATP
l
Y.
fructose 6 P
13=2.8
IATP Aspartate
l
AMP
{/} fructose 6 P
n=1.3 ,..I
ATCase
PF K
Comparison of the models proposed to account [o1" the regulatory properties of E. coli aspartate transcarbamylase (ATCase) and yeast phospho#uctokinase (PFK). These models are quoted from references 2 and 4. (a). ATCase. This representation describes only a functional catalytic and regulatory unit defined as two catalytic chains (one from each catalytic subunit) linked together through a regulatory dimer. Squares and circles correspond respectively to the constrained (T) and relaxed (R) conformations of the catalytic chains, which are involved in the homotropic co-operative interactions between the catalytic sites ; n refers to the experimentally determined Hill number ; 6.8 and 8.2 are the experimentally determined values of the optimum pH of the constrained and relaxed conformations. CTP is inhibitor and ATP activator. (b). PFK. T and R conformations are indicated as such. ATP is inhibitor and AMP activator.
lyzing the different reactions of a metabolic pathway evolved from distinct polypeptides, each catalyzing one reaction, to multienzymatic complexes BIOCHIMIE, 1981, 63, n ° 2.
tive or negative interactions between the regulatory sites, as observed in rabbit muscle phosphofructokinase or glycogen phosphorylase.
A general basic feature of regulatory e n z y m e s . Some general features of the altosteric enzymes will p r o b a b l y emerge in the coming years from the extensive study of a series of these regulatory proteins. T h e work of Franqois Seydoux and his collaborators will r e m a i n a n i m p o r t a n t c o n t r i b u t i o n to this progress. REFERENCES. 1. Laurent, M., Chaffotte, A. F., Tenu, J. P., Roucous, C. & Seydoux, F. (1978) Biochem. Biophys. Res. Commun., 80, 646-652. 2. Lanrent, M., Seydoux, F. & Dessen, P. (1979) J. Biol. Chem., 254, 7515-7520. 3. Nissler, X., Kessler, R., Schellenberger, W. & Hofmann, E. (1979) Biochem. Biophys. Res. Commun., 91, 1462-1467. 4. Thiry, L. & Herv6, G. (1978) J. Mol. Biol., 125, 515534. 5. Gerhart, J. & Schachman, H. (1965) Biochemistry, 4, 1054-1062. 6. Tijane, N. N., Seydoux, F., Hill, M., Roucous, C. & Laurent, M. (1979) FEBS Letters, 105, 249-253.
BIOCH1M1E, 1981, 63, n ° 2.
105
7. Kopperschtager, G., Usbeck, E. & Hofmann, E. (1976) Biochem. Biophys. Res. Commun., 71, 371378. 8. Plietz, P., Damaschun, G., Kopperschlager, G. & Muller, J. J. (1978) FEBS Letters, 91, 230-232. 9. Gerhart, J. C. (1970) Curr. Top. Cell. Regul., 2, 275325. 10. Jacobson, G. R. & Stark, G. R. (1973) In < (Boyer, P. D . e d . ) vol. 9, Part B ; pp. 225-308, Academic Press, London and New York. 11. Kerbiriou, D. 8~ Herv6, G. (1972) J. Mol. Biol., 64, 379-392. 12. Kerbiriou, D. & Herv6, G. (1973) J. Mol. Biol., 78, 687-702. 13. Kerbiriou, D., Herv6, G. & Griffin, J. H. (1977) J. Biol. Chem., 252, 2881-2890. 14. Kantrowitz, E. R., Jacobsberg, L B., Landfear, S. M. & Lipscomb, W. N. (1977) Proc. Natl. Sci. USA, 74, 111-114. 15. Chan, W. W. & Enns, C. A. (1979) Can. J. Biochem., 57, 798-805. 16. Lad, P. M. & Hammes, G. G. (1974) Biochemistry, 13, 4530-4536. t7. Wang, J. H. & Tu, J. I. (1970) J. Biol. Chem., 245, 176-182.
Is the association of concerted homotropic cooperative interactions and local heterotropic effects a general basic feature of regulatory enzymes ? Guy HERVt~.
Laboratoire d'Enzymologie, C.N.R.S., 91190 Gi[-sur-Yvette.
This article is dedicated to the memory of our friend and skillful biochemist Francois Seydoux.
Frangois Seydoux and his collaborators showed that phosphofructokinase from Saccharomyces cerevisiae is made of the association of catalytic and regulatory subunits [1]. From kinetic and spectroscopic studies of the binding of substrates and effectors these authors proposed a model associating concerted homotropic interactions and local heterotropic effects [2]. The same conclusion was reached by Nissler et aI. [3]. Independently a similar model has been proposed to account for the regulatory properties of Escherichia coli aspartate transcarbamylase [4] which is also constituted of catalytic and regulatory subunits [5]. This similarity might be of more general significance. It has been established that yeast phosphofructokinase is an octamer of two different kinds of polypeptides [1, 6, 7, 8] which were characterized as catalytic and regulatory chains on the basis of their AMP and ATP binding properties [1]. The conformational changes induced in the enzyme by these nucleotides and by the substrate fructose-6-phosphate were investigated by initial rate measurements and UV absorption or fluorescence difference spectroscopy. Fructose-6-phosphate appears to bind cooperatively to the catalytic sites of the enzyme and it is shown that this process involves a concerted transition. The effects of the activator AMP and of the inhibitor ATP appear not to involve co-operative interactions and it is concluded that these effectors act through a <~local site by site >>mechanism [2]. The inter-dependence between the effects of these two nucleotides lead the authors to conclude that these metabolites are shifting a single equilibrium in a reverse way. These conclusions are illustrated in the accompanying scheme. Since the inhibitory effect of ATP cannot be completely reversed by AMP it is still possible that the two nuc!eotides provoke distinct local conformational changes ; this possibility is illustrated in the model by dotted arrows (M. Laurent, personal communication).
Escherichia
coli
aspartate
transcarbamylase,
first enzyme of the pyrimidine pathway is made of the association of two catalytic trimers and three regulatory dimers. Catalytic and regulatory chains are different polypeptides. The structural and regulatory properties of this enzyme which shows homotropic and heterotropic interactions have been extensively reviewed [4, 9, 10]. The homotropic co-operative interactions between its catalytic sites for aspartate binding are interpreted in terms of a concerted transition. The catalytic activity is feedback inhibited by CTP and stimulated by ATP. In order to explain the properties of 2T'hioU-ATCase, (a modified form of the enzyme in which the homotropic co-operative interactions between the catalytic sites are selectively abolished) [11-14], and some characteristics of the process of stimulation of the normal enzyme activity by ATP [4] a model has been proposed. This model (represented in the accompanying scheme) associates a concerted transition corresponding to the homotropic co-operative interactions between the catalytic sites with <, local site by site ~>conformational changes corresponding to the action of the effectors ATP and CTP [4]. This interpretation was recently confirmed by the results of a series of elegant experiments showing that the T and R states of the enzyme are still sensitive to the action of ATP and CTP after being <~frozen ,> by means of cross-linking reagents which abolish the concerted transitions [15]. The models proposed account also for the influence of the effectors on the co-operativity between the catalytic sites, since their primary effect on the affinity of the catalytic sites for the substrate will change the degree of saturation of these sites and thus have a secondary effect on the T ~ R equilibrium via the substrate. The striking similarity in behaviour between yeast phosphofructokinase and E. coli aspartate transcarbamylase sug-
104
G. Hervd.
gests that the combination of concerted homotropic interactions and local heterotropic effects might be a general basic feature of the regulatory enzymes made of catalytic and regulatory subunits. Even in the case of enzymes in which the catalytic and regulatory sites are borne by the same polypeptidic chain it has been reported that the use of cross-linking reagents which aboslishes the co-operative interactions does not lead to the disappearance of the heterotropic effects between catalytic and regulatory sites. This is the case of rabbit muscle phosphofructokinase [16] and glycogen phosphorylase [17]. This phenomenon might be of significance concerning the Evolution of proteins. It appears that in a general way the polypeptidic structures cataCONCERTED
made of the aggregation of several of these polypeptides. More integrated multifunctional proteins then appeared, in which the catalytic sites are carried by the same polypeptide chain. Most probably the regulatory properties of protein evolved in the same way from structures resulting from the association of catalytic and regulatory subunits, to proteins in which catalytic and regulatory sites are borne by the same polypeptiJe chain. It seems that the association of catalytic and regulatory chains results in the possibibility of a concerted transition, the effect of which can be modulated by local conformational changes induced by effectors. The more integrated structures, in which catalytic and regulatory sites are formed by the same polypeptide chain, probably allows more complex interactions to occur, including homotropic posiTRANSITIONS fructose S.P
Aspartate I--,
n-
4.7
I
CTP
I.I.I I--
Aspartat~
::ATP
ATP
l
Y.
fructose 6 P
13=2.8
IATP Aspartate
l
AMP
{/} fructose 6 P
n=1.3 ,..I
ATCase
PF K
Comparison of the models proposed to account [o1" the regulatory properties of E. coli aspartate transcarbamylase (ATCase) and yeast phospho#uctokinase (PFK). These models are quoted from references 2 and 4. (a). ATCase. This representation describes only a functional catalytic and regulatory unit defined as two catalytic chains (one from each catalytic subunit) linked together through a regulatory dimer. Squares and circles correspond respectively to the constrained (T) and relaxed (R) conformations of the catalytic chains, which are involved in the homotropic co-operative interactions between the catalytic sites ; n refers to the experimentally determined Hill number ; 6.8 and 8.2 are the experimentally determined values of the optimum pH of the constrained and relaxed conformations. CTP is inhibitor and ATP activator. (b). PFK. T and R conformations are indicated as such. ATP is inhibitor and AMP activator.
lyzing the different reactions of a metabolic pathway evolved from distinct polypeptides, each catalyzing one reaction, to multienzymatic complexes BIOCHIMIE, 1981, 63, n ° 2.
tive or negative interactions between the regulatory sites, as observed in rabbit muscle phosphofructokinase or glycogen phosphorylase.
A general basic feature of regulatory e n z y m e s . Some general features of the altosteric enzymes will p r o b a b l y emerge in the coming years from the extensive study of a series of these regulatory proteins. T h e work of Franqois Seydoux and his collaborators will r e m a i n a n i m p o r t a n t c o n t r i b u t i o n to this progress. REFERENCES. 1. Laurent, M., Chaffotte, A. F., Tenu, J. P., Roucous, C. & Seydoux, F. (1978) Biochem. Biophys. Res. Commun., 80, 646-652. 2. Lanrent, M., Seydoux, F. & Dessen, P. (1979) J. Biol. Chem., 254, 7515-7520. 3. Nissler, X., Kessler, R., Schellenberger, W. & Hofmann, E. (1979) Biochem. Biophys. Res. Commun., 91, 1462-1467. 4. Thiry, L. & Herv6, G. (1978) J. Mol. Biol., 125, 515534. 5. Gerhart, J. & Schachman, H. (1965) Biochemistry, 4, 1054-1062. 6. Tijane, N. N., Seydoux, F., Hill, M., Roucous, C. & Laurent, M. (1979) FEBS Letters, 105, 249-253.
BIOCH1M1E, 1981, 63, n ° 2.
105
7. Kopperschtager, G., Usbeck, E. & Hofmann, E. (1976) Biochem. Biophys. Res. Commun., 71, 371378. 8. Plietz, P., Damaschun, G., Kopperschlager, G. & Muller, J. J. (1978) FEBS Letters, 91, 230-232. 9. Gerhart, J. C. (1970) Curr. Top. Cell. Regul., 2, 275325. 10. Jacobson, G. R. & Stark, G. R. (1973) In <