Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 130-144). Also available on the Internet.

For this pseudoquadreactant enzymatic reaction (Mn2+ is a psuedoreactant), initial velocity patterns were obtained under conditions in which two substrates were maintained saturating while one reactant was varied at several fixed concentrations of the other.

Phosphoenolpyruvate carboxylase (orthophosphate: oxaloacetic acid carboxylyase, E.C.4.1.2.31) was isolated from 4-day germinated peanut cotyledons and purified about 2400-fold. A large-scale purification procedure has been developed in order to obtain large quantities of the highly-purified enzyme. Two assay systems were employed for studying phosphoenolpyruvate carboxylase. Since oxaloacetic acid, the phosphoenolpyruvate carboxylation product, is unstable under the reaction conditions, malate dehydrogenase and reduced nicotinamide-adenine dinucleotide were added in both assays to convert oxaloacetic acid to malic acid, which is stable. One assay method involves the measurement of the incorporation of C¹⁴-bicarbonate into malic acid and the other spectrophotometric determination of the rate of reduced nicotinamide-adenine dinucleotide oxidation. The pH optimum was found to be between 7.8 and 8.3. The sedimentation properties of the enzyme were studied by analytical ultracentrifugation and sucrose density-gradient centrifugation. The sedimentation coefficient (s_20,w) was found to be 13.8S. The Michaelis constants (Km) for Mg⁺⁺ were 4.0x10⁻⁴M and 2.7x10⁻⁴M and for PEP were 6.3xl0⁻⁴M and 5.1x10⁻⁴M as determined by C¹⁴-bicarbonate fixation and the spectrophotometric assay methods, respectively. The Km value for bicarbonate anion was 3.1x10⁻³M using the spectrophotometric assay method. No significant inhibition of the carboxylation reaction was caused by key intermediates in the glycolytic pathway and the Krebs cycle. Although citrate and pyrophosphate inhibited the reaction, these inhibitions were readily reversed by additional Mg⁺⁺ in the reaction mixture. A low concentration of hydroxylamine caused no inhibition of the reaction, but rather had a stabilizing effect on the oxaloacetate formed. Higher concentrations of hydroxylamine produced some inhibition of the reaction. It was, however, concluded that the inhibition was nonspecific. The possibility of the formation of an anhydride linkage between substrates or substrate and enzyme was provisionally ruled out. Trinitrobenzenesulfonic acid and diisopropylfluorophosphate were poor inhibitors of the reaction. Secondary, rather than the primary effects of these compounds on the enzyme were suggested. Thus it is suggested that neither free amino groups nor Βeryl-hydroxyl groups of the enzyme protein participate as active sites. A possible mechanism of the action of phosphoenolpyruvate carboxylase was proposed. That bicarbonate anion is the active attacking species rather than carbon dioxide was proved by conducting the carboxylase-catalyzed carboxylation of phosphoenolpyruvate using O¹⁸-labeled bicarbonate as substrate. Orthophosphate and malate were isolated and analyzed for O¹⁸. The presence of O¹⁸ in orthophosphate was explained as a direct transfer from O¹⁸ bicarbonate. The carboxylation of phosphoenolpyruvate, therefore, appears to involve the participation of the bicarbonate anion as a nucleophilic reagent, its attack on the phosphoryl phosphorus atom of phosphoenolpyruvate, and the formation of a pentacovalent phosphorus in the transition state. The carbonyl character of bicarbonate-derived carbon atom in the transition state being greatly increased would be more susceptible to nucleophilic attack. An intramolecular nucleophilic displacement on this carbon by methylene carbon or phosphoenolpyruvate would result in the formation of a new carbon-carbon bond and cleavage of the carbon-oxygen bond completing the transfer of one bicarbonate oxygen atom to the phosphorus atom. Simultaneous cleavage of the phosphorus-oxygen bond would lead to the formation of the keto-form of oxaloacetic acid and orthophosphate. / Ph. D.

Call number: LD2668 .T4 1985 J54 / Master of Science

Ascaris suum NAD-malic enzyme catalyzes the decarboxylation of oxalacetate and reduction of pyruvate. Thus, the present classification (E.C. 1.1.1.39) for this enzyme should be changed to E.C. 1.1.1.38. 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.

This thesis presents evidence for a novel mechanism of regulation of pyruvate dehydrogenase (PDH) kinase by fatty acids and also results of a study of muscle triacylglycerol concentration. In animals regulation of PDH complex activity is central to the selection of respiratory fuels and to the conservation of glucose during carbohydrate deprivation. The principal means of regulation of PDH complex is interconversion of phosphorylated (inactive) and dephosphorylated (active) forms effected by PDH kinase and PDH phosphatase. Earlier in vitro studies by others had identified both shorter term (min) and longer term (hours) mechanisms of activation of PDH kinase by fatty acid. In the present study PDH kinase activity (as measured by rates of ATP-dependent inactivation of PDH complex in extracts) was shown to be increased when rat heart mitochondria were incubated with palmitoyl-L-carnitine [PC] (and other CoA utilising respiratory substrates). The activation of PDH kinase persisted through removal of respiratory substrate following incubation with CCCP. A comparable effect of PC was also demonstrable in heart mitochondria from 48h-starved rats (i.e. the mechanism may be distinct from that which increases PDH kinase activity in starvation). Rates of ATP-dependent inactivation of PDH complex were also increased when extracts of rat heart mitochondria were incubated with palmitoyl-CoA (PCoA); the increase was comparable with that seen on incubation of intact mitochondria with PC. The PC effect in intact mitochondria and the PCoA effect in mitochondrial extracts may not be identical as PCoA further increased PDH kinase activity in extracts from mitochondria incubated with PC. Rates of incorporation of ³²P from [γ-<sup>32</supP]ATP into PDH complex were unaltered by pnor incubation of mitochondria with PC or by pnor incubation of mitochondrial extracts with PCoA. Three lines of evidence confirmed that the effect of PC to accelerate ATP-dependent inactivation involved phosphorylation of the PDH complex (viz; use of a non-phosphorylatmg ATP analogue; use of known inhibitors of PDH kinase; and use of known activators/inhibitors of PDH phosphatase). Earlier studies had shown that phosphorylation in punfied bovine and porcine PDH complexes is half site (involves only one α-chain in E1 (α2β2) and had suggested that phosphorylation in rat heart complex may be full site (i.e. involves both α-chains). The present study suggests the possibility that elevation of fatty acyl CoA under slaughter house conditions might be a determinant of half site phosphorylation. A method was developed and evaluated for measurement of triacylglycerol in rat soleus muscle strips with the object of investigating factors that may regulate triacylglycerol synthesis in this muscle. This study was abandoned because, although the method was highly reproducible, great variation was found in the triacylglycerol concentration of individual muscles suggesting the possibility of variable contamination with small amounts of adipose tissue.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2004. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Duke University, 2008. / Includes bibliographical references.

Thesis (M.S.)--Brock University, 2009. / Includes bibliographical references (leaves 69-85).