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Pyrimidine Salvage Enzymes in Microorganisms: Labyrinths of Enzymatic Diversity

Description: Pyrimidine salvage pathways are essential to all cells. They provide a balance of RNA synthesis with the biosynthetic pathway in pyrimidine prototrophs and supply all the pyrimidine requirements in auxotrophs. While the pyrimidine biosynthetic pathway is found in almost all organisms and is nearly identical throughout nature, the salvage pathway often differs from species to species, with aspects of salvage seen in every organism. Thus significant taxonomic value may be ascribed to the salvage pathway. The pyrimidine salvage pathways were studied in 55 microorganisms. Nine different salvage motifs, grouped I-IX, were identified in this study based on the presence of different combinations of the following enzymes: cytidine deaminase (Cdd), cytosine deaminase (Cod), uridine phosphorylase (Udp), uracil phosphoribosyltransferase (Upp), uridine hydrolase (Udh), nucleoside hydrolase (Nuh), uridine/cytidine kinase (Udk), 5'-nucleotidase and CMP kinase (Cmk).
Date: December 1995
Creator: Beck, Debrah A. (Debrah Ann)
Partner: UNT Libraries

pH Dependence of the Kinetic Parameters for the Oxalacetate Decarboxylation and Pyruvate Reduction Reactions Catalyzed by Malic Enzyme

Description: Ascaris suum NAD-malic enzyme catalyzes the decarboxylation of oxalacetate and reduction of pyruvate. Thus, the present classification (E.C. for this enzyme should be changed to E.C. In the absence of nucleotide, both the chicken liver NADP-malic enzyme and Ascaris suum NAD-malic enzymes catalyze the decarboxylation of oxalacetate. A study of the pH dependence of kinetic parameters for oxalacetate decarboxylation and pyruvate reduction was carried out for the NAD(P)-malic enzyme with Mg^2+ and Mn^2+ in the presence and absence of nucleotide. In all cases, an enzyme residue is required in its protonated form for reaction while for oxalacetate decarboxylation the β-carboxyl of oxalacetate is required unprotonated. Of a number of inhibitory binding analogs of malate tested, oxalate is the tightest binding inhibitor for Ascaris suum enzyme.
Date: August 1985
Creator: Park, Sang-Hoon
Partner: UNT Libraries

The Effects of Hypothermia on the Release of Cardiac Enzymes

Description: The myocardium is known to release CPK, LDH1 , and GOT in response to ischemia as a result of myocardial infarction. This study was designed to induce the release of cardiac enzymes without adversely effecting the myocardium by perfusion hypothermia, thereby suggesting that these enzymes are not as specific in the diagnosis of myocardial infarction as once thought. Hypothermia was by in vivo perfusion of the left anterior descending coronary artery. Enzyme activity was measured from sera samples spectrophotometrically and electrophoretically. Significant CPK and LDH1 increases were observed in animals perfused between 25 and 19 C. These results indicate that, while heart function remained unchanged, an alteration occurred in the membrane integrity of the myocardial cells.
Date: August 1977
Creator: Strawn, William B.
Partner: UNT Libraries

Exoprotease Production by Aeromonas hydrophila in a Chemically Defined Medium

Description: Wretlind, Heden, and Wadstrom found ammonium sulfate to be inhibitory for the formation of extracellular protease in Aeromonas hydrophila grown in Brain Heart Infusion medium. They demonstrated by manipulating the iron and zinc content within their medium that it is possible to differentially affect the accumulation of hemolysin and protease by A. hydrophila grown in batch culture. Further manipulation of the composition of this medium was done in the present study to determine the effect of other components on the production of protease. The purpose of this study was to determine the factors affecting the level of A. hydrophila protease produced in a chemically defined medium.
Date: May 1985
Creator: Anderson, Paulette S. (Paulette Sue), 1952-
Partner: UNT Libraries

Human phosphoglucose isomerase: isolation and characterization of wild type and the Singh allozyme

Description: A procedure was developed for the rapid isolation of human phosphoglucose isomerase by substrate-induced elution from cellulose phosphate. The high degree of selectivity of the elution provided homogenous enzyme from erythrocytes after a purification of approximately 30,000-fold with a recovery of approximately 70%. The enzyme was also isolated from other human tissues by a similar procedure.
Date: August 1974
Creator: Tilley, Bill E.
Partner: UNT Libraries

Final progress report for DOE grant [Protein dynamics and biocatalysis]

Description: The purpose of this project was to develop and apply large-scale computer simulation methods to enzyme reactions and protein dynamics. New approaches based on the QM/MM methodology were formulated. New insights on the reaction mechanisms of triosephosphate isomerase and chorismate mutase were obtained.
Date: September 3, 2001
Creator: Karplus, Martin
Partner: UNT Libraries Government Documents Department

Site Directed Mutagenesis Of Dienelactone Hydrolase

Description: The role of individual amino acid residues of the enzyme dienelactone hydrolase was investigated. Using the polymerase chain reaction (PCR), a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHI to EcoRI clcD subclone in the plasmid pUC19. Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. Specifically modified proteins from mutated clcD genes (Arg 81 to alanine, Tyr 85 to phenylalanine and Arg 206 to alanine), were encoded by the mutant clones. Enzyme assays showed that dienelactone hydrolase activity of the mutants Arg 81 and Arg 206 was totally abolished. The DLHase enzyme activity of mutant Tyr 85 is greatly decreased by approximately two thirds.
Date: December 1992
Creator: Chen, Wei, 1965-
Partner: UNT Libraries

Studies on the Purification and Phosphorylation of Phosphofructokinase from Ascaris suum

Description: A new procedure has been developed to concentrate the phosphofructokinase from muscle of Ascaris suum with minimum loss of activity. By utilizing this method, 50 ml fraction was concentrated to a final volume of 3 ml in about 1.5 h without loss in enzyme activity. The concentrated enzyme had a specific activity of 64 units per mg. Ascaris muscle-cuticle was incubated in 50 1M solutions of either acetylcholine, serotonin, y-aminobutyric acid, levamisole, or saline alone. Phosphate analysis of the isolated phosphofructokinase from each incubation revealed that the enzyme contained the following moles of phosphate per subunit: 2.9 (acetylcholine), 2.2 (serotonin), 2.0 (y-aminobutyric acid), 1.5 (levamisole), and 3.4 (salne alone). The present study did not establish a direct correlation between degree of phosphorylation and phosphofructokinase activity. Phosphofructokinase from muscle of Ascaris suum appears to contain several phosphorylation sites, and one of these sites is required to be phosphorylated in order for the enzyme to exhibit maximum activity under physiological conditions.
Date: August 1983
Creator: Kaeini, Mohammad R. (Mohammad Reza)
Partner: UNT Libraries

Improving Enzyme Activity and Broadening Selectivity for Biological Desulfurization and Upgrading of Petroleum Feedstocks

Description: The objective of this project was to develop improved biocatalysts for desulfurization and upgrading of petroleum feedstocks. The goal was to improve the activity and broaden the selectivity of desulfurization enzymes using directed evolution as a tool as well as to explore the impact of ring-opening on biological desulfurization
Date: May 12, 2003
Creator: Borole, Abhijeet P.; Hamilton, Choo Y.; Miller, Karen; Davison, Brian; Grossman, Matthew & Shong, Robert
Partner: UNT Libraries Government Documents Department

Studies of methylglyoxal synthase: the distribution of enzyme and chemical mechanism of catalysis

Description: Methylgloxal synthase, which catalyzes the conversion of dihydroxyacetone phosphate to methylglyoxal and inorganic phosphate, has been found in several Enterobacteriaceae. The enzyme along with glyoxalase I and II and D-lactate oxidase, therefore, constitute a nonphosphorylated shunt of the normal glycolytic pathway
Date: May 1978
Creator: Yuan, Pau-Miau
Partner: UNT Libraries

Aza Cope Rearrangement of Propargyl Enammonium Cations Catalyzed By a Self-Assembled `Nanozyme

Description: The tetrahedral [Ga{sub 4}L{sub 6}]{sup 12-} assembly (L = N,N-bis(2,3-dihydroxybenzoyl)-1,5-diaminonaphthalene) encapsulates a variety of cations, including propargyl enammonium cations capable of undergoing the aza Cope rearrangement. For propargyl enammonium substrates that are encapsulated in the [Ga{sub 4}L{sub 6}]{sup 12-} assembly, rate accelerations of up to 184 are observed when compared to the background reaction. After rearrangement, the product iminium ion is released into solution and hydrolyzed allowing for catalytic turnover. The activation parameters for the catalyzed and uncatalyzed reaction were determined, revealing that a lowered entropy of activation is responsible for the observed rate enhancements. The catalyzed reaction exhibits saturation kinetics; the rate data obey the Michaelis-Menten model of enzyme kinetics, and competitive inhibition using a non-reactive guest has been demonstrated.
Date: February 27, 2008
Creator: Hastings, Courntey J.; Fiedler, Dorothea; Bergman, Robert G. & Raymond, Kenneth N.
Partner: UNT Libraries Government Documents Department

Kinetic and Chemical Mechanism of O-Acetylserine Sulfhydrylase-B from Salmonella Typhimurium

Description: Initial velocity studies of O-acetylserine sulfhydrylase-B (OASS-B) from Salmonella typhimurium using both natural and alternative substrates suggest a Bi Bi ping pong kinetic mechanism with double substrate competitive inhibition. The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition. Product inhibition by acetate is S-parabolic noncompetitive, indication of a combination of acetate with E followed by OAS. These data suggest some randomness to the OASS-B kinetic mechanism. The pH dependence of kinetic parameters was determined in order to obtain information on the acid-base chemical mechanism for the OASS-B reaction. A mechanism is proposed in which an enzyme general base accepts a proton from α-amine of O-acetylserine, while a second enzyme general base acts by polarizing the acetyl carbonyl assisting in the β-elimination of the acetyl group of O-acetylserine. The ε-amine of the active site lysine acts as a general base to abstract the α-proton in the β-elimination of acetate. At the end of the first half reaction the ε-amine of the active site lysine that formed the internal Schiff base and the general base are protonated. The resulting α-aminoacrylate intermediate undergoes a Michael addition with HS‾ and the active site lysine donates its proton to the α-carbon to give cysteine and regenerate enzyme to start the second half reaction. In addition, substrate specificity, stereochemistry of the internal Schiff base at C4', and sequence around active site lysine of O-acetylserine sulfhydrylase-A have been determined. The [4'-^3H]pyridoxamine generated by reduction of the internal Schiff base with sodium [^3H]borohydride retained most of its tritium after incubation with apoaspartate aminotransferase. These results agree with the hypothesis put forth by Dunathan (Dunathan, 1971; Dunathan and Voet, 1974) that a single surface (Re face) of the active site PLP is accessible to solvent. The sequence around the active site ...
Date: August 1993
Creator: Tai, Chia-Hui
Partner: UNT Libraries

How Do Enzymes Wear Out? Effects of Posttranslational Modifications on Structure and Stability of Proteins; The Triosephosphate Isomerase Model

Description: Triosephosphate isomerase (EC, TPI) undergoes specific posttranslational modifications (deamidation and oxidation) which are believed to initiate protein turnover by destabilization of the dimer. The crystal structures, amino acid sequences, and aging related changes of TPI from various species have been independently characterized by several laboratories. TPI has thus become the prototype enzyme for examining the initial steps in protein turnover. The binding of substrate enhances the specific deamidation of the mammalian enzyme, and a general mechanism of 'molecular wear and tear' [Gracy, R. W., Yiiksel, K. 0., Chapman, M. L., and Dimitrijevich, S. D. (1990) in Isozymes-Structure, Function and Use in Biology and Medicine (Ogita, Z-I., and Markert, C. L., Eds) pp. 787-817, Wiley-Liss, New York] has been proposed to explain how enzymes may wear out.
Date: December 1991
Creator: Sun, An-Qiang
Partner: UNT Libraries

Measurement of Feedback Inhibition In Vivo and Selection of ATCase Feedback Altered Mutants in Salmonella typhimurium

Description: Aspartate transcarbamoylase (ATCase; encoded by pyrBI genes) is one of the most studied regulatory enzymes in bacteria. It is feedback inhibited by cytidine triphosphate (CTP) and activated by adenosine triphosphate (ATP). Much is known about the catalytic site of the enzyme, not nearly as much about the regulatory site, to which CTP binds. Until now a positive selection for feedback-modified mutants was not available. The selection we have developed involves the use of a pyrA deletion in S. typhimurium. This strain lacks carbamoylphosphate and requires both a pyrimidine and arginine for growth. In this strain citrulline is used to satisfy the pyrimidine and arginine requirements. The minimal flow through the pyrimidine pathway from the citrulline-produced carbamoylphosphate is exquisitely sensitive to feedback control of ATCase by CTP. By elevating the CTP pool, via exogenous cytidine, in a strain that also contains a cytidine deaminase mutant (cdd) growth can be stopped completely, indicating 100% inhibition. It was therefore possible to measure in vivo feedback inhibition of ATCase among the citrulline users and to isolate a family of ATCase regulatory mutants with either modified or no response to effectors.
Date: August 1989
Creator: Bailey, Andrea J., 1952-
Partner: UNT Libraries