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T W Traut

Publications and source records attributed to T W Traut.

At least 37 records · Page 2Linked to original sources
PubMed1988

Enzymes of nucleotide metabolism: the significance of subunit size and polymer size for biological function and regulatory properties.

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.

Enzymes↗
PubMed1987

Regulation of N-carbamoyl-beta-alanine amidohydrolase, the terminal enzyme in pyrimidine catabolism, by ligand-induced change in polymerization.

N-Carbamoyl-beta-alanine (NC beta A) amidohydrolase (EC 3.5.1.6) is regulated in opposing fashion by the substrate, NC beta A and the product, beta-alanine. The native enzyme from rat liver has a molecular weight of 235,000 in the absence of ligands. NC beta A and substrate analogs (N-amidino-beta-alanine, N-carbamoyl-glycine) produced association of the enzyme. beta-Alanine and its analog gamma-aminobutyrate caused dissociation of the enzyme and produced inhibition. Negative cooperativity was observed for the binding of all ligands as measured by the change in polymerization of the enzyme, with an average Hill coefficient (napp) of 0.5. Enzyme that had been dissociated by preincubation with beta-alanine had little or no initial activity; only after a lag of 9 s was a steady state progress curve evident. The existence of a regulatory site is proposed as a model to explain physical and kinetic data. The enzyme activity was highest in rat liver and detectable in kidney; activity was not detected in brain, lung, muscle, or spleen of rat, nor in mouse Ehrlich ascites tumor cells. The rat liver enzyme has a pH optimum of 6.8, with a Km of 6.5 microM for NC beta A and a Ki of 1.08 mM for beta-alanine at this pH.

Amidohydrolases↗
PubMed1987

Uridine kinase: altered subunit size or enzyme expression as a function of cell type, growth stimulation, or mutagenesis.

Using antibody prepared against pure uridine kinase from Ehrlich ascites cells, we have measured the expression of enzyme protein by the Western blot technique. Variations were observed in the Mr of the enzyme subunit for uridine kinase from different species: 32,000 (mouse Ehrlich ascites cells), 30,000 (normal human lymphocytes), 28,000 (mouse tissues), 27,500 (rat tissues). For different normal tissues from the same species, there was no significant variation in the subunit size. Transformed human and mouse cell lines, selected for a deficiency of uridine kinase activity in the presence of inhibitors activated by this enzyme, expressed two cross-reacting proteins, one with a normal (30,000) and one with a smaller (21,000) subunit molecular weight than was found in the parental cell line (human lymphoma), or only a smaller protein of Mr 25,000 (mouse lymphoma). Our results show that selection protocols using metabolite inhibitors do not always repress the expression of the enzyme but instead may lead to selection of those cells that have a mutation in the uridine kinase gene, resulting in the expression of an inactive enzyme. The expression of uridine kinase protein changes when cells are stimulated to divide. For both mouse fibroblasts and human lymphocytes, expression of uridine kinase protein as well as activity clearly increased after cells were stimulated to grow. In fibroblasts, increases are seen by 3 hr after stimulation, and plateau after 9 hr at a sevenfold increase. In lymphocytes, no change is seen until 12 hr after stimulation, and a plateau is not reached until 72 hr, with a total increase of approximately 50-fold. There has been considerable interest in the possibility of uridine kinase isozymes. Except for cells that have been mutagenized, the present results show that, as judged by subunit molecular weight, there appears to be only one enzyme form in normal and neoplastic cells or in cells in which uridine kinase activity is induced.

Animals↗
PubMed1987

Synthesis of hybrid bisnucleoside 5',5"'-P1,P4-tetraphosphates by aminoacyl-tRNA synthetases.

Aminoacyl tRNA synthetases, by means of a back reaction, are able to synthesize certain 5', 5"'-P1, P4-bisnucleoside tetraphosphates of biological importance, such as Ap4A. Here it is shown that HisRS and TrpRS (Bacillus stearothermophilus) and AlaRS (E. coli) also synthesize the hybrid compounds Ap4G, Ap4C, and Ap4U. GlnRS (E. coli) is unable to synthesize any of the above compounds. AlaRS synthesizes Ap4U very poorly, and Ap4C and Ap4G almost as effectively as Ap4A. HisRS and TrpRS synthesize Ap4G, Ap4U and Ap3U quite effectively, and Ap4C very poorly. The fact that hybrid bisnucleoside tetraphosphates can be made by the same enzymes, and at rates comparable to Ap4A, suggests that these compounds may also occur in vivo.

Adenine Nucleotides↗
PubMed1986

Regulation of uridine kinase. Evidence for a regulatory site.

Uridine kinase from mouse Ehrlich ascites tumor cells may exist at 4 degrees C in multiple aggregation states that only slowly equilibrate with one another. Increasing the temperature leads to dissociation, and the appearance of a single predominant species: at 22 degrees C the enzyme exists as a tetramer. There is also a break in the dependence of enzyme activity on temperature as measured in an Arrhenius plot. The feedback inhibitors CTP and UTP cause the enzyme to dissociate to the monomer, whereas the substrate ATP reverses this process. Kinetic studies show that the monomer has little or no activity. Studies of the reaction mechanism show that binding of substrates is ordered, leading to a ternary complex, and release of products is ordered: uridine is the first substrate bound, ADP the first product released. Except for the inhibitors UTP and CTP, all other nucleoside triphosphates, whether purine or pyrimidine, or containing ribose or deoxyribose, act as phosphate donor. Especially interesting are the opposite effects of CTP and dCTP on uridine kinase: unlike CTP, dCTP does not dissociate the enzyme and is competent as a phosphate donor. We propose that the various effects of different ligands are best explained by the existence of a regulatory site (with more stringent specificity than the catalytic site) that controls dissociation of uridine kinase to the inactive monomer.

Adenosine Triphosphate↗
PubMed1986

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 enzyme's biological role.

Base Sequence↗
PubMed1986

Homogeneous uridine kinase from Ehrlich ascites tumor: substrate specificity and inhibition by bisubstrate analogs.

Uridine kinase has been purified to homogeneity from Ehrlich ascites tumor cells. For the phosphate acceptor site, the enzyme shows substrate specificity only for ribopyrimidine nucleosides and is active with various analogs that have limited structural alterations; both endocyclic and exocyclic substituents can be acceptable. Of nucleosides that have been used in the chemotherapy of cancer, 5-fluorouridine, 6-azauridine, and 3-deazauridine are good substrates, whereas arabinosylcytosine is a poor substrate. No analogs are better substrates than the physiological substrates uridine and cytidine. 5', 5''' -P1, P4-Bisnucleoside oligophosphate bisubstrate analogs (e.g., Ap4U, Ap5U) were synthesized and tested as inhibitors. The most effective compound was Ap4U; with a Ki of 197 microM, it bound more tightly than ATP but no better than uridine. Ap3A, Ap4A, and Ap5A were also tested, with the result that both Ap4A and Ap4U were most effective, suggesting that this size of bisubstrate analog most closely approaches the spacing of the catalytic site.

Adenine Nucleotides↗
PubMed1985

Uridine kinase from Ehrlich ascites carcinoma. Purification and properties of homogeneous enzyme.

Uridine kinase from Ehrlich ascites tumor cells has been purified about 60,000-fold to apparent homogeneity and with an overall recovery of about 40%. This purification was achieved using phosphocellulose and adenosine 5'-triphosphate-agarose affinity chromatography. The subunit molecular mass as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 31,000 daltons. With two-dimensional electrophoresis, only one spot was observed, indicating the absence of isoenzymes. Multiple peaks of activity are routinely observed on ion exchange chromatography or gel filtration, for both crude preparations or homogeneous uridine kinase, in agreement with our earlier results that this enzyme exists as multiple interconvertible oligomeric forms (Payne, R. C., and Traut, T. W. (1982) J. Biol. Chem. 257, 12485-12488). The purified enzyme has a specific activity of 283 mumol/min/mg of protein at 22 degrees C. Initial velocity studies using uridine and ATP are consistent with a sequential mechanism. Km values for uridine, cytidine, and ATP are 40, 57, and 450 microM, respectively. CTP and UTP are competitive inhibitors with respect to ATP, with Ki values for CTP and UTP of 10 and 61 microM, respectively. The enzyme was active with several nucleoside analogs, the Km values being 69 microM (5-fluorouridine), 200 microM (3-deazauridine), and 340 microM (6-azauridine). The pure enzyme is very sensitive to freezing, but can be maintained at O degrees C for 8 weeks with only 20% loss of activity. For long-term storage, enzyme in 50% glycerol can be maintained at -20 degrees C for many months with no detectable loss of activity.

Animals↗
PubMed1985

A Salmonella typhimurium strain defective in uracil catabolism and beta-alanine synthesis.

A selection procedure for uracil catabolism mutant strains involving indicator dye plates was developed. Using this method, a strain defective in uracil catabolism has been isolated in Salmonella typhimurium that was temperature-sensitive at 42 degrees C where it required low concentrations of N-carbamoyl-beta-alanine, beta-alanine or pantothenic acid for growth. An extract of the mutant strain degraded uracil at 37 degrees C at a significantly diminished rate compared to that observed for the wild-type strain under the same growth conditions. The conversion of dihydrouracil to N-carbamoyl-beta-alanine was blocked at all temperatures examined in the mutant strain. By means of genetic analysis, the mutant strain was determined to be defective at two genetic loci. Transduction studies with bacteriophage P22 indicated that the panD gene is mutated in this strain, accounting for its beta-alanine requirement. Episomal transfers between Escherichia coli and the mutant strain provided evidence that the defect in uracil catabolism was located in another region of the S. typhimurium chromosome.

Alanine↗
PubMed1984

Pyrimidine catabolism: individual characterization of the three sequential enzymes with a new assay.

We have developed a one-dimensional thin-layer chromatography procedure that resolves the initial substrate uracil and its catabolic derivatives dihydrouracil, N-carbamoyl-beta-alanine (NCBA) and beta-alanine. This separation scheme also simplifies the preparation of the radioisotopes of N-carbamoyl-beta-alanine and dihydrouracil. Combined, these methods make it possible to assay easily and unambiguously, jointly or individually, all three enzyme activities of uracil catabolism: dihydropyrimidine dehydrogenase, dihydropyrimidinase, and N-carbamoyl-beta-alanine amidohydrolase. Earlier reports had presented data suggesting that these three enzyme activities were combined in a complex because they appeared to be controlled at a single genetic locus [Dagg, C. P., Coleman, D.L., & Fraser, G.M. (1964) Genetics 49, 979-989] and because they appeared able to channel metabolites [Barrett, H.W., Munavalli, S.N., & Newmark, P. (1964) Biochim. Biophys. Acta 91, 199-204]. Although the three enzymes from rat liver have similar sizes, with apparent molecular weights of 218 000 for dihydropyrimidine dehydrogenase, 226 000 for dihydropyrimidinase, and 234 000 for NC beta A amidohydrolase, they are easily separated from each other. Kinetic studies show no evidence of substrate channeling and therefore do not support a model for an enzyme complex. The earlier reports may be explained by our studies on the amidohydrolase, which suggest that under certain conditions this enzyme may become the rate-limiting step in uracil catabolism.

Amidohydrolases↗
PubMed1982

Regulation of uridine kinase quaternary structure. Dissociation by the inhibitor CTP.

Uridine kinase from mouse Ehrlich ascites cells can exist in a variety of different aggregation states, from monomer up to aggregates that may contain 32 or more subunits. With very crude enzyme preparations, uridine kinase activity is always associated with several different coexisting molecular weight species. Changes in the aggregation state are produced in the presence of normal effectors (orthophosphate, ATP and CTP) at physiological concentrations. With uridine kinase that has been purified 9,000-fold, enzyme activity is associated with only a single molecular weight species, but is still responsive to the same physiological effectors. In the presence of orthophosphate, uridine kinase has a molecular weight of 380,000 (appropriate for a dodecamer). In the presence of CTP, the enzyme dissociates with concomitant loss of activity. The dissociated enzyme can be reassociated to the native size. These results imply that alteration of the enzyme's quaternary structure by normal effectors constitutes a mechanism for regulating uridine kinase activity in vivo.

Animals↗
PubMed1980

Dependence of the aggregation and conformation states of uridine 5'-phosphate synthase on pyrimidine nucleotides. Evidence for a regulatory site.

Uridine 5'-phosphate (UMP) synthase is multifunctional protein that contains the last two enzyme activities of the de novo pathway for UMP biosynthesis, orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate (OMP) decarboxylase (EC 4.1.1.23). We have previously reported that UMP synthase from mouse Ehrlich ascites cells can exist in at least three distinct aggregation and/or conformation states, as measured by changes in sedimentation through sucrose gradients [Traut, T. W., & Jones, M. E. (1979) J. Biol. Chem, 254, 1143-1150]. The major sedimenting species were a 3.6S monomer, a 5.1S dimer, and a 5.6S species. The formation of the 5.1S dimer from the 3.6S monomer occurs in the presence of ligands that are competitive inhibitors at the OMP decarboxylase catalytic site. This paper presents evidence for a regulatory site, distinct from either of the two catalytic sites, which appears to mediate the conversion of the 5.1S dimer to the 5.6S form upon binding certain pyrimidine nucleotides (OMP, UMP, and 6-azaUMP). Since UMP synthase sediments predominantly as a dimer in the presence of substrates, regulation of the aggregation/conformation state of this multifunctional protein may be physiologically significant.

Binding Sites↗
PubMed1980

Dependence of the catalytic activities on the aggregation and conformation states of uridine 5'-phosphate synthase.

Uridine 5'-phosphate (UMP) synthase is a multifunctional protein that contains the last two enzyme activities for the de novo biosynthesis of UMP, orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate (OMP) decarboxylase (EC 4.1.1.23). The native enzyme from mouse Ehrlich ascites cells exists in at least three distinct aggregation/conformation states as measured by sedimentation in sucrose gradients: a 3.6S monomer, a 5.1S dimer, and a conformationally altered 5.6S dimer. It has previously been reported that a variety of ligands (of which the most effective is OMP) mediate the conversion of the 3.6S monomer to the two types of dimers. Initial velocity studies with the enzyme in the different native states show that all three forms of UMP synthase have phosphoribosyltransferase activity but that the OMP decarboxylase is either uniquely or at least predominantly associated with the 5.6S form. Activation of this enzyme activity by the substrate appears to be the result of both a dimerization and a conformation step.

Animals↗