Abstracts of Communications 568th Meeting of the Biochemical Society held jointly with Sveriges Biokemiska Forening and the British Biophysical Society University of Aberdeen, 23, 24 and 25 March 1977
SUBUNIT INTERACTION AND ENZYME REGULATION : a Colloquium organized on behalf of the Biochemical Society and Sveriges Biokemiska Forening by J. Jeffery (Aberdeen) and B. Mannervik (Stockholm) Chairman’s Introductory Remarks HAMISH M. KEIR Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen AB9 lAS, Scotland, U.K.
This colloquium on subunit interactions in enzymes brings together several aspects of modern enzymology including enzyme kinetics, tertiary- and quaternary-structural studies and transmission of specific signals between the subunits of oligomeric enzymes. The study of proteins at the level of tertiary and quaternary structures has progressed substantially since the early days of the work of Kendrew and Perutz and their colleagues on myoglobin and haemoglobin. Nevertheless, despite a rapid expansion of research, notably within recent years on enzymes, there are but few examples of comprehensive and incisive achievements comparable with the establishment of the structurefunction relationships for the haemoglobins. X-ray-diffraction studies of protein interactions (Liljas & Rossman, 1974) have, of course, been productive to an extent, and have presented an insight into the train of events that extends from the amino acid sequences encoded in DNA to the h l tertiary or quaternary structures of proteins. For instance, there are now known to be primary structures which are rather different from each other, but which assume essentially the same tertiary structure, as in the myoglobins and the a- and p-chains of the haemoglobins. Recurring patterns (domains) have been identified in the tertiary structures of certain groups of proteins. A co-ordinated programme of research on the glycolytic enzymes and some dehydrogenases has given clear indications of tertiary relationships among several of them, particularly in the nucleotide-binding sites (domains) of lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase, alcohol dehydrogenaseand phosphoglyceratekinase (cf. Blake, 1975). Such studies allow development of hypotheses on the evolution of protein structure, but have not thus far revealed the precise details of the mechanism of the action of the enzymes or the functional significance of subunit interaction and the specificity thereof. Nevertheless, structural analyses of enzymes, of enzymesubstrate, enzyme-substrate-analogue, enzyme-inhibitor and enzyme-co-factor complexes have the potential to yield valuable information on the locations of active sites and allosteric regulatory sites,
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BIOCHEMICAL SOCIETY TRANSACTIONS
as well as the nature of eiTector-induced conformational changes. These aspects will be discussed in this colloquium with respect to alcohol dehydrogenase and phosphorylase 6 . The detailed functional relationships between and among the monomers and protomers of an oligomeric enzyme are in most cases poorly understood, but quite spectacular advances have been made in a few specific instances, for example, aspartate carbamoyltransferase (Jacobson & Stark, 1973). Much information is available on negative and positive effector molecules, on the types of protomer and monomer in this oligomeric regulatory enzyme, on the arrangement of the subunits in the enzyme and about the kinetic responses of the enzyme to substrate in the presence and absence of the effectors. Moreover, the enzyme may be dissociated to and reconstituted from its component subunits in vitro. Kinetic studies have revealed much on the mechanism of action of allosteric enzymes, but often, as will be described below, no clear relationship can be established between such studies (together with structural data) and subunit interaction. A remarkable example will be given in this colloquium of the binding of substrates and effectors only to one of the two non-identical subunits in an enzyme in which the active site is formed by both subunits. Equally remarkable are proteins comprised of subunits whose amino acid sequences are encoded in different genomes. One such protein, the DNA polymerase induced by bacteriophage T7, will be described in this colloquium. Another is the RNA replicase of bacteriophage Q/3 (Clark, 1974). This enzyme comprises four subunits, only one of which (subunit 11) is encoded in the viral RNA. The other three are encoded in the DNA of the bacterial host. Subunit I is identical with, and may be replaced by, an interference factor (i) which inhibits protein biosynthesis at the stage of initiation (Groner et ul., 1972). Subunits I11 and IV are identical with the elongation factors (in protein biosynthesis) EF-Tu and EF-Ts respectively (Blumenthal et al., 1972). This subunit inteiaction, of course, links protein biosynthesis with RNA biosynthesis. The technique of protein complementation (restoration in vivo or in vitro of enzyme activity by non-covalent interaction of different proteins or polypeptides) may be applied to oligomeric regulatory enzymes to determine the number and identity of subunits in them and the functions of different regions of polypeptide chains. Tertiary and quaternary structures also may be investigated by protein complementation (Zabin & Villarejo, 1975). For example glutamate dehydrogenase of Neurospora crussu has been studied with this technique by Fincham and co-workers. Subunit interactions in this oligomeric enzyme (six identical subunits, each of mol.wt. 50000) suggest a conformational correction of a mutant protein with altered tertiary structure when it is hybridized with a mutant protein which is inactive because of amino acid substitution in the active site (Codtiington & Fincham, 1965; Sundaram & Fincham, 1968). Clearly, protein-complementation studies have the potential to test models of subunit interaction in regulatory enzymes. The well-known sequential (Koshland, 1970) and symmetry (Monod et uf., 1965) models for co-operative interactions in oligomeric enzymes, attempt to relate quaternary structure to the dynamics of allosteric regulation. Protein complementation together with X-ray analysis may well be applicable to the testing of such models. Whatever, co-operative interactions of the subunits of oligomeric enzymes are highly specific, and this aspect will also be considered in the first paper of the colloquium. Blake, C. C. F. (1975) Ess~zysBiochem. 11, 37-39 Blumenthal,T.,Landers,T.A. & Weber, K. (1972)Proc. Nutl. Acud. Sci. U.S.A. 69,1313-1317 Clark, B. F. C. (1974) in Co,mpunionto Biochemistry (Bull, A. T., Lagnado, J. R., Thomas, J. 0. & Tipton, K. F., eds.), pp. 1-86, Longman, London Coddington, A. &. Fincham, J. R. S . (1965)J. Mol. Biol. 12, 152-161 Groner, Y., Pollack, Y.,Berissi, H. & Revel, M. (1972)Nature (Lond.)239, 16-19 Jacobson, G. R. & Stark, (3. R. (1973) Enzymes 3rd Ed. 9,225-308
1977
568th MEETING. ABERDEEN
605
Koshland, D. E. (1970) Enzymes 3rd. Ed. 1, 341-396 Liljas, A. & Rossman, M. G. (1974) Annu. Rev. Biochem. 43.475-507 Monod, I., Wyman, J. & Changeux, J.-P.(1965)J.Mol. Biol. 12,88-118 Sundaram, T. K. & Fincham, J. R. S. (1968) J. Bacteriol. 95, 787-792 Zabin, I. & Villarejo, M. R. (1975) Annu. Rev. Biochem. 44, 295-313
The Specificity of Subunit Interactions D. E. KOSHLAND, JR. Department of Biochemistry, University of California, Berkeley, CA 94120, U.S.A.
The joining together of subunits requires selectivity at the same level of evolutionary discrimination as that required for active sites themselves. The evidence for this is the failure of proteins to hybridize incorrectly either in vivo or in vitro when deliberate mixing experiments were performed with proteins prepared from different species or by mixtures of proteins from the same species. Subunits associated correctly to produce recognizable enzymes, with incorrect hybridization being an extremely minor factor. In view of the large number of associationswhich are possible, these results indicatea high degree of selectivity in subunit interactions. The purpose of these interactions is in most cases to transmit information from one subunit to the other. Many dissociated proteins are unstable or inactive, indicating that the association of the subunits leads to a stabilization or an activation process. In these cases the individual subunits are acting as allosteric effects of each other, leading to situations in which the monomer is inactive and the dimer is active. However, since many monomeric enzymes are active, it seems quite clear that the structure of a subunit could be designed to produce activity. Hence the purpose of subunit association would seem most likely to be transmission of energy and information, although this cannot be the exclusive function of oligomeric proteins. The polymerization of individual peptide chains can occur in two ways: (a) to create a situation of symmetry in all the subunits, and (b) to create a situation of asymmetry, an ad-type of association. X-ray crystallography has revealed that both types exist, at least in the crystal, and it is therefore of interest to examine their role in catalytic action. Three possibilities would seem to exist. (a) The reactivity of the proteins is indicated by their X-ray structure; i.e. very fundamental differences in reactivity will be observed between the a- and a’-subunits. (b) The differencesin subunit structures are so minor energetically that the reactivities of the subunits are essentially equal even though there are observable differences in three-dimensional space. (c) The reactivity of amino acid residues involves such subtle changes in shape that subunits appearing to be identical nevertheless can have very significant differences in reactivity. To study the nature of subunit interaction, glyceraldehyde 3-phosphate dehydrogenase was examined for its reactivity with a variety of alkylating agents. Although each of these agents, forming a covalent link with the cysteine-149 residue, produces half of the site’s reactivity, the change in reactivity of neighbouring subunits has subtle effects which depend on the structure of the reagent. It is not sufficient to explain these results to say that only two pre-exisitng structures can exist, or even that two states of the protein of different conformational natures are in equilibrium with each other. The interactionsare far too complex and too specific. If, in addition, the further permutations of NAD-induced changes are included, the specificity of the interactions is even more apparent. To determine the contribution of pre-existing asymmetry, a re-ordered alkylation experiment was performed. The protein was treated with dinitrofluorobenzene until two of the four subunits were completely alkylated. This produces inactive protein. Such a result would be predicted either by preferential reaction with the two reactive
Vol. 5
SUBUNIT INTERACTION AND ENZYME REGULATION : a Colloquium organized on behalf of the Biochemical Society and Sveriges Biokemiska Forening by J. Jeffery (Aberdeen) and B. Mannervik (Stockholm) Chairman’s Introductory Remarks HAMISH M. KEIR Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen AB9 lAS, Scotland, U.K.
This colloquium on subunit interactions in enzymes brings together several aspects of modern enzymology including enzyme kinetics, tertiary- and quaternary-structural studies and transmission of specific signals between the subunits of oligomeric enzymes. The study of proteins at the level of tertiary and quaternary structures has progressed substantially since the early days of the work of Kendrew and Perutz and their colleagues on myoglobin and haemoglobin. Nevertheless, despite a rapid expansion of research, notably within recent years on enzymes, there are but few examples of comprehensive and incisive achievements comparable with the establishment of the structurefunction relationships for the haemoglobins. X-ray-diffraction studies of protein interactions (Liljas & Rossman, 1974) have, of course, been productive to an extent, and have presented an insight into the train of events that extends from the amino acid sequences encoded in DNA to the h l tertiary or quaternary structures of proteins. For instance, there are now known to be primary structures which are rather different from each other, but which assume essentially the same tertiary structure, as in the myoglobins and the a- and p-chains of the haemoglobins. Recurring patterns (domains) have been identified in the tertiary structures of certain groups of proteins. A co-ordinated programme of research on the glycolytic enzymes and some dehydrogenases has given clear indications of tertiary relationships among several of them, particularly in the nucleotide-binding sites (domains) of lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase, alcohol dehydrogenaseand phosphoglyceratekinase (cf. Blake, 1975). Such studies allow development of hypotheses on the evolution of protein structure, but have not thus far revealed the precise details of the mechanism of the action of the enzymes or the functional significance of subunit interaction and the specificity thereof. Nevertheless, structural analyses of enzymes, of enzymesubstrate, enzyme-substrate-analogue, enzyme-inhibitor and enzyme-co-factor complexes have the potential to yield valuable information on the locations of active sites and allosteric regulatory sites,
Vol. 5
604
BIOCHEMICAL SOCIETY TRANSACTIONS
as well as the nature of eiTector-induced conformational changes. These aspects will be discussed in this colloquium with respect to alcohol dehydrogenase and phosphorylase 6 . The detailed functional relationships between and among the monomers and protomers of an oligomeric enzyme are in most cases poorly understood, but quite spectacular advances have been made in a few specific instances, for example, aspartate carbamoyltransferase (Jacobson & Stark, 1973). Much information is available on negative and positive effector molecules, on the types of protomer and monomer in this oligomeric regulatory enzyme, on the arrangement of the subunits in the enzyme and about the kinetic responses of the enzyme to substrate in the presence and absence of the effectors. Moreover, the enzyme may be dissociated to and reconstituted from its component subunits in vitro. Kinetic studies have revealed much on the mechanism of action of allosteric enzymes, but often, as will be described below, no clear relationship can be established between such studies (together with structural data) and subunit interaction. A remarkable example will be given in this colloquium of the binding of substrates and effectors only to one of the two non-identical subunits in an enzyme in which the active site is formed by both subunits. Equally remarkable are proteins comprised of subunits whose amino acid sequences are encoded in different genomes. One such protein, the DNA polymerase induced by bacteriophage T7, will be described in this colloquium. Another is the RNA replicase of bacteriophage Q/3 (Clark, 1974). This enzyme comprises four subunits, only one of which (subunit 11) is encoded in the viral RNA. The other three are encoded in the DNA of the bacterial host. Subunit I is identical with, and may be replaced by, an interference factor (i) which inhibits protein biosynthesis at the stage of initiation (Groner et ul., 1972). Subunits I11 and IV are identical with the elongation factors (in protein biosynthesis) EF-Tu and EF-Ts respectively (Blumenthal et al., 1972). This subunit inteiaction, of course, links protein biosynthesis with RNA biosynthesis. The technique of protein complementation (restoration in vivo or in vitro of enzyme activity by non-covalent interaction of different proteins or polypeptides) may be applied to oligomeric regulatory enzymes to determine the number and identity of subunits in them and the functions of different regions of polypeptide chains. Tertiary and quaternary structures also may be investigated by protein complementation (Zabin & Villarejo, 1975). For example glutamate dehydrogenase of Neurospora crussu has been studied with this technique by Fincham and co-workers. Subunit interactions in this oligomeric enzyme (six identical subunits, each of mol.wt. 50000) suggest a conformational correction of a mutant protein with altered tertiary structure when it is hybridized with a mutant protein which is inactive because of amino acid substitution in the active site (Codtiington & Fincham, 1965; Sundaram & Fincham, 1968). Clearly, protein-complementation studies have the potential to test models of subunit interaction in regulatory enzymes. The well-known sequential (Koshland, 1970) and symmetry (Monod et uf., 1965) models for co-operative interactions in oligomeric enzymes, attempt to relate quaternary structure to the dynamics of allosteric regulation. Protein complementation together with X-ray analysis may well be applicable to the testing of such models. Whatever, co-operative interactions of the subunits of oligomeric enzymes are highly specific, and this aspect will also be considered in the first paper of the colloquium. Blake, C. C. F. (1975) Ess~zysBiochem. 11, 37-39 Blumenthal,T.,Landers,T.A. & Weber, K. (1972)Proc. Nutl. Acud. Sci. U.S.A. 69,1313-1317 Clark, B. F. C. (1974) in Co,mpunionto Biochemistry (Bull, A. T., Lagnado, J. R., Thomas, J. 0. & Tipton, K. F., eds.), pp. 1-86, Longman, London Coddington, A. &. Fincham, J. R. S . (1965)J. Mol. Biol. 12, 152-161 Groner, Y., Pollack, Y.,Berissi, H. & Revel, M. (1972)Nature (Lond.)239, 16-19 Jacobson, G. R. & Stark, (3. R. (1973) Enzymes 3rd Ed. 9,225-308
1977
568th MEETING. ABERDEEN
605
Koshland, D. E. (1970) Enzymes 3rd. Ed. 1, 341-396 Liljas, A. & Rossman, M. G. (1974) Annu. Rev. Biochem. 43.475-507 Monod, I., Wyman, J. & Changeux, J.-P.(1965)J.Mol. Biol. 12,88-118 Sundaram, T. K. & Fincham, J. R. S. (1968) J. Bacteriol. 95, 787-792 Zabin, I. & Villarejo, M. R. (1975) Annu. Rev. Biochem. 44, 295-313
The Specificity of Subunit Interactions D. E. KOSHLAND, JR. Department of Biochemistry, University of California, Berkeley, CA 94120, U.S.A.
The joining together of subunits requires selectivity at the same level of evolutionary discrimination as that required for active sites themselves. The evidence for this is the failure of proteins to hybridize incorrectly either in vivo or in vitro when deliberate mixing experiments were performed with proteins prepared from different species or by mixtures of proteins from the same species. Subunits associated correctly to produce recognizable enzymes, with incorrect hybridization being an extremely minor factor. In view of the large number of associationswhich are possible, these results indicatea high degree of selectivity in subunit interactions. The purpose of these interactions is in most cases to transmit information from one subunit to the other. Many dissociated proteins are unstable or inactive, indicating that the association of the subunits leads to a stabilization or an activation process. In these cases the individual subunits are acting as allosteric effects of each other, leading to situations in which the monomer is inactive and the dimer is active. However, since many monomeric enzymes are active, it seems quite clear that the structure of a subunit could be designed to produce activity. Hence the purpose of subunit association would seem most likely to be transmission of energy and information, although this cannot be the exclusive function of oligomeric proteins. The polymerization of individual peptide chains can occur in two ways: (a) to create a situation of symmetry in all the subunits, and (b) to create a situation of asymmetry, an ad-type of association. X-ray crystallography has revealed that both types exist, at least in the crystal, and it is therefore of interest to examine their role in catalytic action. Three possibilities would seem to exist. (a) The reactivity of the proteins is indicated by their X-ray structure; i.e. very fundamental differences in reactivity will be observed between the a- and a’-subunits. (b) The differencesin subunit structures are so minor energetically that the reactivities of the subunits are essentially equal even though there are observable differences in three-dimensional space. (c) The reactivity of amino acid residues involves such subtle changes in shape that subunits appearing to be identical nevertheless can have very significant differences in reactivity. To study the nature of subunit interaction, glyceraldehyde 3-phosphate dehydrogenase was examined for its reactivity with a variety of alkylating agents. Although each of these agents, forming a covalent link with the cysteine-149 residue, produces half of the site’s reactivity, the change in reactivity of neighbouring subunits has subtle effects which depend on the structure of the reagent. It is not sufficient to explain these results to say that only two pre-exisitng structures can exist, or even that two states of the protein of different conformational natures are in equilibrium with each other. The interactionsare far too complex and too specific. If, in addition, the further permutations of NAD-induced changes are included, the specificity of the interactions is even more apparent. To determine the contribution of pre-existing asymmetry, a re-ordered alkylation experiment was performed. The protein was treated with dinitrofluorobenzene until two of the four subunits were completely alkylated. This produces inactive protein. Such a result would be predicted either by preferential reaction with the two reactive
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