viii, 173 leaves : ill. + appendix / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1971) from the Dept. of Biochemistry, University of Adelaide

Thesis (Ph.D. 1971) from the Dept. of Bio-chemistry, University of Adelaide.

viii, 135 leaves : ill., bibliog. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1972

Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1972.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Escherichia coli phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxylase (transphorsphorylating) EC 4.1.1.49) catalyzes the decarboxylation and subsequent phosphorylation of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the presence of Mg2+ATP and synergistic catalysis has been observed in the presence of Ca2+ or Mn2+. Structural analyses have shown that active site residues Arg333, Ser250 and Tyr207 are coordinated differently in E. coli PCK structures complexed with Mg2+ATP-oxalate, Mg2+ATP-Mn2+-pyruvate and Mg2+ATP-Ca2+-pyruvate; hence, we hypothesize that the function of Arg333, Ser250 and Tyr207, depends on the absence or presence of Ca2+ or Mn2+ during catalysis by E. coli phosphoenolpyruvate carboxykinase (PCK). In order to verify this hypothesis, site directed mutagenesis of the pckA gene was used to convert Arg333 to Gln, Ser250 to Ala and Tyr207 to Phe, while 14CO2 exchange assay and x-ray crystallography were used to determine the effects of these mutations on catalysis by E. coli PCK in the presence of OAA and Mg2+ATP with Ca2+ or Mn2+ metal ions. Kinetic analysis showed that the Tyr207Phe mutation decrease kcat by 1.7 fold, while Ser250Ala and Arg333Gln reduced kcat by 10.8 and 4,555 fold respectively in the presence of Mg2+ATP and OAA. In the presence of Mg2+ATP, OAA and Ca2+, Arg333Gln, Ser250Ala and Tyr207Phe mutations reduced kcat by 11,688, 44 and 2 fold respectively. In the presence of Mg2+ATP, OAA and Mn2+ Arg333Gln, Ser250Ala and Tyr207Phe mutations reduced kcat by 2,880, 4 and 5.5 fold respectively. The crystal structure of Ser250Ala complexed with Mg2+ATP-Mn2+-pyruvate, showed that in the presence of Mn2+, Ser250Ala mutation reduced the angle between the γ-phosphate of ATP and residue 250 by 6.2 Å and increased the distance between the hydroxyl group of Tyr207 and the CH2 group of pyruvate by 0.5 Å. As a result we conclude that Arg333 is important for oxaloacetate decarboxylation and phosphorylation. During catalysis in the presence of Mg2+ATP with or without Ca2+ or Mn2+, Ser250 functions to maintain one γ-phosphate oxygen of ATP in an eclipsed conformation, while Tyr207 functions to drive oxaloacetate decarboxylation during catalysis in the presence of Mn2+ ion. Kinetic and structural studies of E. coli PCK have previously been used to show that Asp269 is involved in metal coordination, while Lys254 and Arg65 are important for Mg2+ATP and OAA binding to E. coli PCK respectively. In this study the E. coli PCK Asp269Asn-Mg2+ATP-Ca2+-pyruvate crystal structure showed that the Asp269Asn mutation reduced the number of ligands coordinating Ca2+ from seven to three, while no electron density was observed for Mg2+ATP and OAA in Lys254Ser and Arg65Gln crystal structures respectively.

No description available.

Phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step of gluconeogenesis, and the gene which encodes this enzyme (pckA) is transcriptionally regulated. High pckA expression is observed in succinate-grown cells, while little expression is observed in glucose-grown cells. pckA regulatory mutants have previously been isolated (Osteras et al. 1997) and pckR, a gene encoding a Lacl-GaIR DNA-binding transcriptional regulator, has been implicated in the regulation of pckA transcription. Here we shew that pckR insertion mutations result in a dramatic decrease in pckA expression even in succinate-grown cells. We demonstrate that the previously identified rpk-9 mutation is tightly linked to pckR. The rpk-9 mutation results in constitutive pckA expression, and we show that plasmids carrying the pckR gene complement the rpk-9 mutation in glucose-grown cells. A putative Lacl-GaIR operator binding site has been identified in the pckA promoter, however no evidence of an interaction between this site and the pckR gene product could be found. / Thesis / Master of Science (MS)

In cultured rat hepatocytes the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK) is known to be induced by glucagon via an elevation of cAMP. Prostaglandin E₂ has been shown to antagonize the glucagon-activated cAMP formation, glycogen phosphorylase activity and glucose output in hepatocytes. It was the purpose of the current investigation to study the potential of PGE₂ to inhibit the glucagon-induced expression of PCK on the level of mRNA and enzyme activity. PCK mRNA and enzyme activity were increased by 0.1 nM glucagon to a maximum after 2 h and 4 h, respectively. This increase was completely inhibited if 10 μM PGE2 was added concomitantly with glucagon. This inhibition by PGE₂ of glucagon-induced PCK activity was abolished by pertussis toxin treatment. When added at the maximum of PCK mRNA at 2 h, PGE₂ accelerated the decay of mRNA and reduced enzyme activity. This effect was not reversed by pertussis toxin treatment. Since in liver PGE₂ is derived from Kupffer cells, which play a key role in the local inflammatory response, the present data imply that during inflammation PGE₂ may reduce the hepatic gluconeogenic capacity via a Gᵢ-linked signal chain.

No description available.