Investigation of the mechanism of action of pep carboxylase

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.

Name thesis:degree_name

Ph. D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biochemistry and Nutrition

Department dc:contributor.department

Biochemistry and Nutrition

Grantor dc:publisher

Virginia Polytechnic Institute

Year dc:date.issued

1964

Author dc:creator

• Maruyama, Hitoshi

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/10919/101486

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/101486