Thomas W. Traut

Physiological concentrations of purines and pyrimidines.

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.

Dissociation of Enzyme Oligomers: A Mechanism for Allosteric Regulation

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.

Uracil Metabolism—UMP Synthesis from Orotic Acid or Uridine and Conversion of Uracil to β-Alanine: Enzymes and cDNAs

Publisher Summary The importance of maintaining balanced pyrimidine nucleotide metabolism is evident because it has been shown that many biosynthetic pathways are dependent on pyrimidines. Pyrimidine nucleotides are also important for the continued biosynthesis of various macromolecules, such as phospholipids and polysaccharides, for which specific nucleotides are required as cofactors in activating appropriate precursors, as exemplified by CMP-choline and UDP-glucose. Even the conversion of uracil to β-alanine is a biosynthetic pathway, although historically this has been referred to as catabolism. A biosynthetic designation for this pathway comes from the importance of β-alanine, which functions as a neurotransmitter, as well as a building block for various dipeptides. These peptides include the ubiquitous pantothenate, the precursor for the acyl-carrier protein in fatty acid synthase and for coenzyme A. The inability of humans to synthesize pantothenate makes it a vitamin. The biochemical roles of carnosine and other dipeptides containing β-alanine are discussed in the chapter. Nucleotides presumably exist at very low concentrations in the extracellular environment, though no values have been published for their concentration in blood. DNA sequencing suggests that many mammalian or human proteins may be coded by two or more genes, and that such gene duplication may provide a simple safety factor in the event of a mutation in one gene, or provide a more sophisticated regulatory strategy if the genes lead to slightly altered isozymes to be expressed in unique tissues.

[21] Orotate phosphoribosyltransferase: Orotidylate decarboxylase (Ehrlich Ascites Cell)

Publisher Summary This chapter describes purification procedure related to the orotate phosphoribosyltransferase enzyme. Assay procedures reported include methods measuring the spectrophotometric changes that occur when a pyrimidine base is converted to a nucleotide or when orotidylic acid (OMP) is converted to uridylic acid (UMP), methods that separate the free base from the nucleotides, and methods measuring the release of [14C]O2 from [14C]carboxyl-labeled orotate or orotidylate. This assay is appropriate for both crude or pure enzyme preparations. In this procedure orotate, OMP, and UMP are separated from one another by thin-layer chromatography. It is also useful when one wishes to estimate how effective inhibitors of the decarboxylase are when the OMP level is that maintained at a steady-state level by the complex itself,for example, when orotate and phosphoribosyl pyrophosphate (P-Rib-PP) are the substrates and only the OMP formed by the transferase is present, this assay is ideal

Purine nucleoside phosphorylase. Allosteric regulation of a dissociating enzyme.

Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) catalyzes the phosphorolysis of (d)guanosine or inosine to form the base (guanine or hypoxanthine) and (d)ribose-1-P; the enzyme has therefore been defined as catabolic (1,2). The enzyme may also be considered as biosynthetic, since the salvage of purines to form nucleotides is largely via a one step phosphoribosyltransferase reaction. Reports characterizing this enzyme have not been consistent. Based on the native Mr, the enzyme has been reported as a trimer, a dimer, or a monomer (references in #3). Also, some studies have reported negative cooperativity with phosphate (references in #4), or nucleosides (references in #4). Some ambiguity about the above results arose when other studies reported no cooperativity with phosphate, or with nucleosides (references in #4).

Allosteric regulation of purine nucleoside phosphorylase

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).

β-Alanine synthase: Purification and allosteric properties

beta-Alanine synthase has been purified greater than 1000-fold to homogeneity from rat liver. The enzyme has a subunit molecular weight of 42,000 and a native size of hexamer. The enzyme undergoes ligand-induced changes in polymerization: association in response to the substrate, N-carbamoyl-beta-alanine, and the inhibitor, propionate; and dissociation in response to the product, beta-alanine. The ability of the substrate to associate the pure native enzyme to a larger polymeric species was exploited in the final purification step. The purified enzyme had a pI of 6.7, a Km of 8 microM, and a kcat/Km of 7.9 x 10(4) M-1 s-1. Positive cooperativity was observed toward the substrate N-carbamoyl-beta-alanine, with nH = 1.9. Such cooperativity occurred at substrate concentrations below 12 nM, so that this activation most likely occurs at a regulatory site, with a significantly stronger affinity for N-carbamoyl-beta-alanine than that shown by the catalytic site. The enzyme was sensitive to denaturation, which could be minimized by avoiding heat steps during the purification and by the presence of reducing agents. Such denatured enzyme had little change in Vmax, but had much higher Km, and had also lost the ability to associate or dissociate in response to effectors. After purification, enzyme stability was achieved by the addition of glycerol and detergent.

Enzymes of Nucleotide Metabolism: The Significance of Subunit Size and Polymer Size for Biological Function and Regulatory Propertie

The 72 enzymes in nucleotide metabolism, from all sources, have a distribution of subunit sizes similar to those from other surveys: an average subunit Mr of 47,900, and a median size of 33,300. The same enzyme, from whatever source, usually has the same subunit size (there are exceptions); enzymes having a similar activity (e.g., kinases, deaminases) usually have a similar subunit size. Most simple enzymes in all EC classes (except class 6, ligases/synthetases) have subunit sizes of less than 30,000. Since structural domains defined in proteins tend to be in the Mr range of 5,000 to 30,000, it may be that most simple enzymes are formed as single domains. Multifunctional proteins and ligases have subunits generally much larger than Mr 40,000. Analyses of several well-characterized ligases suggest that they also have two or more distinct catalytic sites, and that ligases therefore are also multifunctional proteins, containing two or more domains. Cooperative kinetics and evidence for allosteric regulation are much more frequently associated with larger enzymes: such complex functions are associated with only 19% of enzymes having a subunit Mr less than or equal to 29,000, and with 86% of all enzymes having a subunit Mr greater than 50,000. In general, larger enzymes have more functions. Only 20% of these enzymes appear to be monomers; the rest are homopolymers and rarely are they heteropolymers. Evidence for the reversible dissociation of homopolymers has been found for 15% of the enzymes. Such changes in quaternary structure are usually mediated by appropriate physiological effectors, and this may serve as a mechanism for their regulation between active and less active forms. There is considerable structural organization of the various pathways: 19 enzymes are found in various multifunctional proteins, and 13 enzymes are found in different types of multienzyme complexes.

Significance of the enzyme complex that synthesizes UMP in ehrlich ascites cells

Abstract The de novo biosynthesis of pyrimidine nucleotides is completed by two sequential enzyme activities that convert orotate plus 5-phosphoribosyl-1-pyrophosphate to orotidine-5′-monophosphate (OMP) and PP i and then decarboxylate OMP to produce 5′-uridylic acid. In mammalian cells the two enzyme activities, orotate phosphoribosyltransferase and orotidine-5′-phosphate decarboxylase, form a normally inseparable enzyme complex. It was previously reported that this complex is able to channel the intermediate product, OMP (Traut, T. W., and Jones, M. E., 1977, J. Biol. Chem. 252 , 8374–8381). The studies reported here indicate that one advantage of this channeling of OMP is to spare OMP from being degraded to orotidine by a potentially competitive nucleotidase activity. Yeast cells have two separate enzymes instead of an enzyme complex, and lack the ability to channel OMP. The OMP formed in yeast cells is not degraded because these cells lack significant nucleotidase activity. These results suggest that the capability for channeling OMP may have been important in evolving the enzyme complex found in mammalian cells.

Are proteins made of modules

Analysis of a set of well characterized enzymes shows that the size of a protein subunit is directly related to the number of unique ligand binding functions described for the particular enzyme. The average size increment is about 5 000 Da per ligand binding function. This value corresponds very well to: (a) the amount of polypeptide chain required to form a stable folded structure, and (b) the size of polypeptide coded by the average exon. This reinforces the hypothesis that exon-coded modules are basic architectural units for proteins. Key predictive elements of this hypothesis are: 1) generally each module has a unique function, such as the ability to bind a specific ligand; 2) the size of an enzyme subunit should be determined by the number of modules required to accomplish the enzymes biological role.