The sub-cellular localisation and regulatory properties of pyruvate carboxylase from Rhizopus arrhizus.
Cell-free extracts of Rhizopus arrhizus contain exclusively cytosolic pyruvate carboxylase and NAD-glutamate dehydrogenase, a single mitochondrial isoenzyme of NADP-isocitrate dehydrogenase, and both mitochondrial and cytosolic isoenzymes of NADP-malate dehydrogenase (decarboxylating). Other enzymes examined have sub-cellular localisations similar to those characteristic of mammalian liver. Purified preparations of R. arrhizus pyruvate carboxylase are subject to partial regulatory inhibition by L-aspartate and 2-oxoadipate. L-Glutamate acts as a less effective analogue of L-aspartate while 2-oxoglutarate is ineffective. Competition studies indicate the presence of separate inhibitory sites for L-aspartate and 2-oxoadipate. Under routine assay conditions R. arrhizus pyruvate carboxylase shows significant activation by acyl derivatives of coenzyme A with long chain acyl CoA being more effective than acetyl-CoA. This activation is no longer observed in the presence of high concentrations of pyruvate, MgATP2- and HCO-3. The concentrations of L-aspartate and 2-oxoadipate required to give 50% inhibition ([I]0.5), and the maximal extents of inhibition, are increased by addition of acetyl-CoA. Acetyl-CoA increases the sigmoidal character of the relationship: initial rate/[L-aspartate], but decreases this parameter for the relationship: initial rate/[2-oxoadipate]. The studies indicate that R. arrhizus possesses an entirely cytosolic pathway for the conversion of glucose to fumaric acid and that both the organisation of pyruvate metabolism and the regulation of pyruvate carboxylase differ significantly in this organism as compared to that proposed previously for Aspergillus nidulans.
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000214:Acyl Coenzyme A / Q000494:pharmacology; D000272:Adipates / Q000494:pharmacology; D001224:Aspartic Acid / Q000494:pharmacology; D002498:Centrifugation; D003998:Dicarboxylic Acids / Q000494:pharmacology; D004588:Electrophoresis, Cellulose Acetate; D004789:Enzyme Activation / Q000187:drug effects; D011766:Pyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D012233:Rhizopus / Q000201:enzymology* / Q000254:growth & development; D013347:Subcellular Fractions / Q000201:enzymology*; D014233:Tricarboxylic Acids / Q000494:pharmacology
D000214:Acyl Coenzyme A; D000272:Adipates; D003998:Dicarboxylic Acids; D014233:Tricarboxylic Acids; D001224:Aspartic Acid; D000105:Acetyl Coenzyme A; C001918:alpha-ketoadipic acid; D011766:Pyruvate Carboxylase