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 (μM) are: ATP, 3,152±1,698; GTP, 468±224; UTP, 567±460 and CTP, 278±242. For deoxynucleosidestriphosphate (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 μM. 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 μM; 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.

Physiological concentrations of purines and pyrimidines.

The Arabidopsis genome contains ∼200 genes that encode proteins with similarity to the nucleotide binding site and other domains characteristic of plant resistance proteins. Through a reiterative process of sequence analysis and reannotation, we identified 149 NBS-LRR–encoding genes in the Arabidopsis (ecotype Columbia) genomic sequence. Fifty-six of these genes were corrected from earlier annotations. At least 12 are predicted to be pseudogenes. As described previously, two distinct groups of sequences were identified: those that encoded an N-terminal domain with Toll/Interleukin-1 Receptor homology (TIR-NBS-LRR, or TNL), and those that encoded an N-terminal coiled-coil motif (CC-NBS-LRR, or CNL). The encoded proteins are distinct from the 58 predicted adapter proteins in the previously described TIR-X, TIR-NBS, and CC-NBS groups. Classification based on protein domains, intron positions, sequence conservation, and genome distribution defined four subgroups of CNL proteins, eight subgroups of TNL proteins, and a pair of divergent NL proteins that lack a defined N-terminal motif. CNL proteins generally were encoded in single exons, although two subclasses were identified that contained introns in unique positions. TNL proteins were encoded in modular exons, with conserved intron positions separating distinct protein domains. Conserved motifs were identified in the LRRs of both CNL and TNL proteins. In contrast to CNL proteins, TNL proteins contained large and variable C-terminal domains. The extant distribution and diversity of the NBS-LRR sequences has been generated by extensive duplication and ectopic rearrangements that involved segmental duplications as well as microscale events. The observed diversity of these NBS-LRR proteins indicates the variety of recognition molecules available in an individual genotype to detect diverse biotic challenges.

/pdf/genome-wide-analysis-of-nbs-lrr-encoding-genes-in-5eegxpfsdp.pdf

Genome-Wide Analysis of NBS-LRR–Encoding Genes in Arabidopsis

https://works.bepress.com/steven-whitham/22/download/

The product of the tobacco mosaic virus resistance gene N: Similarity to toll and the interleukin-1 receptor

▪ Abstract In “gene-for-gene” interactions between plants and their pathogens, incompatibility (no disease) requires a dominant or semidominant resistance (R) gene in the plant, and a corresponding avirulence (Avr) gene in the pathogen. Many plant/pathogen interactions are of this type. R genes are presumed to (a) enable plants to detect Avr-gene-specified pathogen molecules, (b) initiate signal transduction to activate defenses, and (c) have the capacity to evolve new R gene specificities rapidly. Isolation of R genes has revealed four main classes of R gene sequences whose products appear to activate a similar range of defense mechanisms. Discovery of the structure of R genes and R gene loci provides insight into R gene function and evolution, and should lead to novel strategies for disease control.

/pdf/plant-disease-resistance-genes-3jj1d7brmf.pdf

Plant Disease Resistance Genes

Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide++ + dihydrochloride] is widely used as a specific inhibitor of the Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) family of protein kinases. This study examined the inhibition mechanism and profile of actions of Y-27632 and a related compound, Y-30141 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(1H-pyrrolo[2, 3-b]pyridin-4-yl)cyclohexan-ecarboxamide dihydrochloride]. Y-27632 and Y-30141 inhibited the kinase activity of both ROCK-I and ROCK-II in vitro, and this inhibition was reversed by ATP in a competitive manner. This suggests that these compounds inhibit the kinases by binding to the catalytic site. Their affinities for ROCK kinases as determined by K(i) values were at least 20 to 30 times higher than those for two other Rho effector kinases, citron kinase and protein kinase PKN. [(3)H]Y-30141 was taken up by cells in a temperature- and time-dependent and saturable manner, and this uptake was competed with unlabeled Y-27632. No concentrated accumulation was found, suggesting that the uptake is a carrier-mediated facilitated diffusion. Y-27632 abolished stress fibers in Swiss 3T3 cells at 10 microM, but the G(1)-S phase transition of the cell cycle and cytokinesis were little affected at this concentration. Y-30141 was 10 times more potent than Y-27632 in inhibiting the kinase activity and stress fiber formation, and it caused significant delay in the G(1)-S transition and inhibition of cytokinesis at 10 microM.

Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases.

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-1 and NMP-2 bind to the pentose and phosphate of the bound ligand. NMP-1 is found in almost all the kinases that phosphorylate AMP, CMP, GMP, dTMP, or UMP. NMP-3 a 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 γ-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.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1994.tb18835.x

The functions and consensus motifs of nine types of peptide segments that form different types of nucleotide‐binding sites

In considering the origin and evolution of proteins, the possibility that proteins evolved from exons coding for specific structure-function modules is attractive for its economy and simplicity but is not systematically supported by the available data. However, the number of correspondences between exons and units of protein structure-function that have so far been identified appears to be greater than expected by chance alone. The available data also show (i) that exons are fairly limited in size but are large enough to specify structure-function modules in proteins; (ii) that the position of introns for homologous domains in the same gene is reasonably stable, but there is also evidence for mechanisms that alter the position or existence of introns; and (iii) that it is possible that the observed relationship of exons to protein structure represents a degenerate state of an ancestral correspondence between exons and structure-function modules in proteins.

Do exons code for structural or functional units in proteins

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...

Dissociation of Enzyme Oligomers: A Mechanism for Allosteric Regulation

SECTION 1. INTRODUCTION TO ENZYMES 1. INTRODUCTION TO ENZYMES 1.1 INTRODUCTION 1.1.1 Why Are Enzymes Needed? 1.1.2 Allosteric Enzymes 1.2 THE STRUCTURES AND CONFORMATIONS OF PROTEINS 1.2.1 Protein Conformations 1.2.2 Protein Structures 1.2.3 Multi-domain Proteins 1.2.3.1 Evolution of Multi-domain Proteins 1.2.3.2 Interaction Between Domains 1.2.3.3 Alternate Oligomer Structures for the Same Enzyme 1.3 NORMAL VALUES FOR CONCENTRATIONS AND RATES 1.3.1 Concentrations of Enzymes 1.3.2 How Fast Are Enzymes? 1.4 BRIEF HISTORY OF ENZYMES 1.5 USEFUL RESOURCES 1.5.1 Websites 1.5.2 Reference Books 1.5.2.1 General Enzymology 1.5.2.2 Allosteric Enzymes 1.5.2.3 Enzyme Kinetics 1.5.2.4 Ligand Binding and Energetics 1.5.2.5 Enzyme Chemistry and Mechanisms 1.5.2.6 Enzymes in Metabolism 1.5.2.7 History of Enzymology 1.5.2.8 Hemoglobin 2. THE LIMITS FOR LIFE DEFINE THE LIMITS FOR ENZYMES 2.1 NATURAL CONSTRAINTS THAT ARE LIMITING 2.1.1 The Possible Concentration of Enzymes is Most Likely to be Limiting 2.1.2 The Rate for Enzymatic Steps Must Be Faster than Natural, but Undesired and Harmful Reactions 2.1.2.1 Oxygen Radicals 2.1.2.2 Metabolic Acidity 2.1.2.3 Ultraviolet Radiation 2.1.3 DNA Modifyng Enzymes: Accuracy Is More Important Than Speed 2.1.4 Signaling Systems: Why Very Slow Rates Can Be Good 2.1.5 What Is the Meaning of the Many Enzymes for Which Slow Rates Have Been Published? 2.2 PARAMETERS FOR BINDING CONSTANTS 2.2.1 The Importance of Being Good Enough 2.2.2 The Range of Binding Constants 2.3 Enzyme specificity: kcat/Km 2.3.1 A Constant kcat/Km May Permit Appropriate Changes for Enzymes With the Same Enzyme Mechanism 2.3.2 The Specificity Constant May Apply to Only One of the Two Substrates for a Group of Enzymes With the Same Mechanism 2.3.3 The Same Enzyme Can Maintain Constant Specificity While Adapting to Changes 2.3.4 The Limits to kcat/Km 2.2.5 Ribozymes and the RNA World? 3. ENZYME KINETICS 3.1 TIME FRAMES FOR MEASURING ENZYME PROPERTIES 3.2 STEADY STATE KINETICS 3.2.1 The Meaning of v and kcat 3.3 The Most Common Graphic Plots 3.3.1 The Michaelis-Menten Plot 3.3.2 The Lineweaver-Burk Plot 3.3.3 The Eadie-Hofstee Plot 3.3.4 The Hill Plot 3.4 Interpreting Binding Constants 3.5 ENERGETICS OF ENZYME REACTIONS 3.5.1 Michaelis-Menten Model 3.5.2 Briggs-Haldane Model 3.5.3 Additional Intermediates Model 4. PROPERTIES AND EVOLUTION OF ALLOSTERIC ENZYMES 4.1 DIFFERENT PROCESSES FOR CONTROLLING THE ACTIVITY OF AN ENZYMATIC REACTION 4.1.1 Modifying the Activity of an Existing Enzyme 4.1.2 Modifying activity by ligand binding 4.1.3 Modifying activity by covalent modification 4.1.4 Modifying activity by altered gene transcription 4.1.5 Modifying activity by proteolysis 4.2 EVOLVING ALLOSTERIC ENZYMES 4.2.1 Allostric Regulation by Stabilizing the Appropriate Species in an Ensemble 4.2.2 Evolution of Allosteric Enzymes 4.3 URACIL PHOPHORIBOSYLTRANSFERASE: DIFFERENT REGULATORY STRATEGIES FOR THE SAME ENZYME 5.

Thomas W. Traut

Papers

Physiological concentrations of purines and pyrimidines.

The functions and consensus motifs of nine types of peptide segments that form different types of nucleotide‐binding sites

Do exons code for structural or functional units in proteins

Dissociation of Enzyme Oligomers: A Mechanism for Allosteric Regulation

Allosteric regulatory enzymes