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
606
BIOCHEMICAL SOCIETY TRANSACTIONS
subunits of a pre-existing asymmetry structure or by the induced distortion of neighbouring subunits in an induced-fit-typemechanism. To distinguish between these cases, the remaining subunits were alkylated under forcing conditions by iodoacetamide, and then the dinitrophenyl groups were removed by thiolation. The di-substituted protein produced in this way was inactive. Clearly it would be expected to be active if the preexisting asymmetry model with no further conformational changes applied, since in that case the initially reactive subunits would have been regenerated by the thiolysis to active protein. Moreover further tests showed that the order of alkylation made no difference; the activity of the protein depended entirely on the number of thiol groups modified. Hence the protein has identical subunits insofar as reactivity is concerned. As yet X-ray crystallography on all forms of glyceraldehyde 3-phosphate dehydrogenase has not been completed. These results from methods utilizing chemical tools do not clearly predict the structure for the reasons outlined above. A pre-existing asymmetry situation could be present, giving a slight preferential reaction of one subunit over the other in the initial alkylation. That would seem unlikely from the re-ordered alkylation results, but it certainly cannot be eliminated. There are many amino acid changes between the a- and ,%subunitsof haemoglobin, and yet the binding of oxygen is quite similar. It would seem logical that two identical proteins with slightly different conformational aspects might have very similar properties and still exist in different shapes in three-dimensional space. Clearly the data indicate that induced conformational changes after the preexisting asymmetry must be a major feature in the properties of this protein. Further, because of the nature of these induced conformational changes, it seems apparent that the symmetry of the completely saturated enzyme has no necessary relationship to asymmetry in the apoenzyme. The subtle nature of induced conformational changes means that ligand-induced effects can also lead to associations or dissociations. A particularly important case of induced association has been found in the receptor interactions in bacterial membranes. Purification and isolation of the galactose and ribose receptors has shown that they compete with each otheir for a common site on the bacterial membrane. In aqueous solution these receptors exist as monomers. By attaching fluorescent labels to these proteins, a ligand-induced conformational change can be observed. Moreover, it is found that this ligand-induced comformational change is essential for the association of the receptor with the components of the signalling system anchored to the membrane. The proteins not only associate with the signalling system, but also compete with each other for this common component, even though they possess little similarity in amino acid sequence and lack cross-reactivitywith antibodies. Clearly a site has been selected on the surface, possibly by convergent evolution, to lead to association with a common configuration in space. This ability to have competition between protein subunits in contrast with direct competition between the sugar residues has considerable advantages to the organism in terms of creating a heirarchy of controls. This function of subunit interaction may be a common feature of sensory systems where such a hierarchy of controls provides survival value to the organism.
Mechanism and Cont:roI of RibonucIeoside Diphosphate Reducfase LARS THELANDER Medical Nobel Institute, Department of Biochemistry, Karolinska Institute, S-104 01 Stockholm, Sweden
When a cell starts to make DNA it needs a balanced supply of the precursors, the four deoxyribonucleoside trijphosphates. These are made by a direct reduction of the corresponding ribonucleotides in a reaction catalysed by the enzyme ribonucleotide reductase (Hogenkamp 6% Sando 1974). The finding that the deoxyribonucleotides are 1977
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
606
BIOCHEMICAL SOCIETY TRANSACTIONS
subunits of a pre-existing asymmetry structure or by the induced distortion of neighbouring subunits in an induced-fit-typemechanism. To distinguish between these cases, the remaining subunits were alkylated under forcing conditions by iodoacetamide, and then the dinitrophenyl groups were removed by thiolation. The di-substituted protein produced in this way was inactive. Clearly it would be expected to be active if the preexisting asymmetry model with no further conformational changes applied, since in that case the initially reactive subunits would have been regenerated by the thiolysis to active protein. Moreover further tests showed that the order of alkylation made no difference; the activity of the protein depended entirely on the number of thiol groups modified. Hence the protein has identical subunits insofar as reactivity is concerned. As yet X-ray crystallography on all forms of glyceraldehyde 3-phosphate dehydrogenase has not been completed. These results from methods utilizing chemical tools do not clearly predict the structure for the reasons outlined above. A pre-existing asymmetry situation could be present, giving a slight preferential reaction of one subunit over the other in the initial alkylation. That would seem unlikely from the re-ordered alkylation results, but it certainly cannot be eliminated. There are many amino acid changes between the a- and ,%subunitsof haemoglobin, and yet the binding of oxygen is quite similar. It would seem logical that two identical proteins with slightly different conformational aspects might have very similar properties and still exist in different shapes in three-dimensional space. Clearly the data indicate that induced conformational changes after the preexisting asymmetry must be a major feature in the properties of this protein. Further, because of the nature of these induced conformational changes, it seems apparent that the symmetry of the completely saturated enzyme has no necessary relationship to asymmetry in the apoenzyme. The subtle nature of induced conformational changes means that ligand-induced effects can also lead to associations or dissociations. A particularly important case of induced association has been found in the receptor interactions in bacterial membranes. Purification and isolation of the galactose and ribose receptors has shown that they compete with each otheir for a common site on the bacterial membrane. In aqueous solution these receptors exist as monomers. By attaching fluorescent labels to these proteins, a ligand-induced conformational change can be observed. Moreover, it is found that this ligand-induced comformational change is essential for the association of the receptor with the components of the signalling system anchored to the membrane. The proteins not only associate with the signalling system, but also compete with each other for this common component, even though they possess little similarity in amino acid sequence and lack cross-reactivitywith antibodies. Clearly a site has been selected on the surface, possibly by convergent evolution, to lead to association with a common configuration in space. This ability to have competition between protein subunits in contrast with direct competition between the sugar residues has considerable advantages to the organism in terms of creating a heirarchy of controls. This function of subunit interaction may be a common feature of sensory systems where such a hierarchy of controls provides survival value to the organism.
Mechanism and Cont:roI of RibonucIeoside Diphosphate Reducfase LARS THELANDER Medical Nobel Institute, Department of Biochemistry, Karolinska Institute, S-104 01 Stockholm, Sweden
When a cell starts to make DNA it needs a balanced supply of the precursors, the four deoxyribonucleoside trijphosphates. These are made by a direct reduction of the corresponding ribonucleotides in a reaction catalysed by the enzyme ribonucleotide reductase (Hogenkamp 6% Sando 1974). The finding that the deoxyribonucleotides are 1977
