The concentrations of bases, nucleosides, and nucleosides mono-, di- and tri-phosphate are compared for about 600 published values. The data are predominantly from mammalian cells and fluids. For the most important ribonucleotides, average concentrations +/- SD (microM) are: ATP, 3,152 +/- 1,698; GTP, 468 +/- 224; UTP, 567 +/- 460 and CTP, 278 +/- 242. For deoxynucleosides-triphosphate (dNTP), the concentrations in dividing cells are: dATP, 24 +/- 22; dGTP, 5.2 +/- 4.5; dCTP, 29 +/- 19 and dTTP 37 +/- 30. By comparison, dUTP is usually about 0.2 microM. For the 4 dNTPs, tumor cells have concentrations of 6-11 fold over normal cells, and for the 4 NTPs, tumor cells also have concentrations 1.2-5 fold over the normal cells. By comparison, the concentrations of NTPs are significantly lower in various types of blood cells. The average concentration of bases and nucleosides in plasma and other extracellular fluids is generally in the range of 0.4-6 microM; these values are usually lower than corresponding intracellular concentrations. For phosphate compounds, average cellular concentrations are: Pi, 4400; ribose-1-P, 55; ribose-5-P, 70 and P-ribose-PP, 9.0. The metal ion magnesium, important for coordinating phosphates in nucleotides, has values (mM) of: free Mg2+, 1.1; complexed-Mg, 8.0. Consideration of experiments on the intracellular compartmentation of nucleotides shows support for this process between the cytoplasm and mitochondria, but not between the cytoplasm and the nucleus.

From an analysis of current data on 16 protein structures with defined nucleotide-binding sites consensus motifs were determined for the peptide segments that form such nucleotide-binding sites. This was done by using the actual residues shown to contact ligands in the different protein structures, plus an additional 50 sequences for various kinases. Three peptide segments are commonly required to form the binding site for ATP or GTP. Binding motif Kinase-1 a is found in almost all sequences examined, and functions in binding the phosphates of the ligand. Variant versions, comparable to Kinase-1 a, are found in a subset of proteins and appear to be related to unique functions of those enzymes. Motif Kinase-2 contains the conserved aspartate that coordinates the metal ion on Mg-ATP. Motif Kinase-3 occurs in at least four versions, and functions in binding the purine base or the pentose. Two protein structures show ATP-binding at a separate regulatory site, formed by the motifs Regulatory-1 and Regulatory-2. Structures for adenylate kinase and guanylate kinase show three different sequence motifs that form the binding site for a nucleoside monophosphate (NMP). NMP-I and NMP-2 bind to the pentose and phosphate of the bound ligand. NMP-I is found in almost all the kinases that phosphorylate AMP, CMP, GMP, dTMP, or UMP. NMP-3a is found in kinases for AMP, GMP, and UMP, while NMP-3b binds only GMP. For the binding of NTPs, three distinct types of nucleotide-binding fold structures have been described. Each structure is associated with a particular function (e.g. transfer of the y-phosphate, or of the adenylate to an acceptor) and also with a particular spatial arrangement of the three Kinase segments evident in the linear sequence for the protein.With the rapid proliferation of DNA sequences in the last decade, the finding that a few consensus sequences might generally correspond to ATP-binding sites was immediately useful. In 1982 Walker et al. deduced from a search of available sequences for ATP-binding proteins that one, or both, of two sequence motifs appeared in nine proteins, with four proteins having both sequences [l]. Since both of these motifs occurred in porcine adenylate kinase, at positions corresponding to the proposed nucleotide-bindmg site in the crystal structure [2], the association of these sequences with actual ATP-bindmg appeared to be established. Although adenylate kinase uses ATP to phosphorylate AMP, the crystal structure then available had some ambiguity as to whether the Walker A site or the Walker B site bound ATP or AMP; the appearance of these motif sequences in other ATP-binding proteins favored the former nucleotide site. Since this original paper also found only a Walker B sequence in phosphofructokinase [l], an easy interpretation was that either

No abstract

Most enzymes exist as oligomers or polymers, and a significant subset of these (perhaps 15% of all enzymes) can reversibly dissociate and reassociate in response to an effector ligand. Such a change in subunit assembly usually is accompanied by a change in enzyme activity, providing a mechanism for regulation. Two models are described for a physical mechanism, leading to a change in activity: (1) catalytic activity depends on subunit conformation, which is modulated by subunit dissociation; and (2) catalytic or regulatory sites are located at subunit interfaces and are disrupted by subunit dissociation. Examples of such enzymes show that both catalytic sites and regulatory sites occur at the junction of 2 subunits. In addition, for 9 enzymes, kinetic studies supported the existence of a separate regulatory site with significantly different affinity for the binding of either a substrate or a product of that enzyme. Over 40 dissociating enzymes are described from 3 major metabolic areas: carbohydrate metabolism, nucleotide metabolism, and amino acid metabolism. Important variables that influence enzyme dissociation include: enzyme concentration, ligand concentration, other cellular proteins, pH, and temperature. All these variables can be readily manipulated in vitro, but normally only the first two are physiological variables. Seven of these enzymes are most active as the dissociated monomer, the others as oligomers, emphasizing the importance of a regulated equilibrium between 2 or more conformational states. Experiments to test whether enzyme dissociation occurs in vivo showed this to be the case in 6 out of 7 studies, with 4 different enzymes.

From an analysis of current data on 16 protein structures with defined nucleotide-binding sites consensus motifs were determined for the peptide segments that form such nucleotide-binding sites. This was done by using the actual residues shown to contact ligands in the different protein structures, plus an additional 50 sequences for various kinases. Three peptide segments are commonly required to form the binding site for ATP or GTP. Binding motif Kinase-1 a is found in almost all sequences examined, and functions in binding the phosphates of the ligand. Variant versions, comparable to Kinase-1 a, are found in a subset of proteins and appear to be related to unique functions of those enzymes. Motif Kinase-2 contains the conserved aspartate that coordinates the metal ion on Mg-ATP. Motif Kinase-3 occurs in at least four versions, and functions in binding the purine base or the pentose. Two protein structures show ATP-binding at a separate regulatory site, formed by the motifs Regulatory-1 and Regulatory-2. Structures for adenylate kinase and guanylate kinase show three different sequence motifs that form the binding site for a nucleoside monophosphate (NMP). NMP-I and NMP-2 bind to the pentose and phosphate of the bound ligand. NMP-I is found in almost all the kinases that phosphorylate AMP, CMP, GMP, dTMP, or UMP. NMP-3a is found in kinases for AMP, GMP, and UMP, while NMP-3b binds only GMP. For the binding of NTPs, three distinct types of nucleotide-binding fold structures have been described. Each structure is associated with a particular function (e.g. transfer of the y-phosphate, or of the adenylate to an acceptor) and also with a particular spatial arrangement of the three Kinase segments evident in the linear sequence for the protein.With the rapid proliferation of DNA sequences in the last decade, the finding that a few consensus sequences might generally correspond to ATP-binding sites was immediately useful. In 1982 Walker et al. deduced from a search of available sequences for ATP-binding proteins that one, or both, of two sequence motifs appeared in nine proteins, with four proteins having both sequences [l]. Since both of these motifs occurred in porcine adenylate kinase, at positions corresponding to the proposed nucleotide-bindmg site in the crystal structure [2], the association of these sequences with actual ATP-bindmg appeared to be established. Although adenylate kinase uses ATP to phosphorylate AMP, the crystal structure then available had some ambiguity as to whether the Walker A site or the Walker B site bound ATP or AMP; the appearance of these motif sequences in other ATP-binding proteins favored the former nucleotide site. Since this original paper also found only a Walker B sequence in phosphofructokinase [l], an easy interpretation was that either