Pyruvate carboxylase from Saccharomyces cerevisiae. Quaternary structure, effects of allosteric ligands and binding of avidin.
The quaternary structure of pyruvate carboxylase purified from Saccharomyces cerevisiae was investigated by electron microscopic examination of negatively stained samples. In the most frequently observed projection form four intensity maxima were arranged at the corners of a rhombus; a cleft along the longitudinal axis of individual protomers could often be discerned. The observation of occasional triangular and dual-intensity projections and the interconversion of all three projection forms in tilting studies indicates that this tetrameric enzyme has a structure very similar to the tetrahedron-like configuration previously proposed for pyruvate carboxylases from vertebrate sources [Mayer, F., Wallace, J. C. and Keech, D. B. (1980) Eur. J. Biochem. 112, 265-272] and Aspergillus nidulans [Osmani, S. A., Mayer, F., Marston, F. A. O., Selmes, I. P. and Scrutton, M. C. (1984) Eur. J. Biochem. 139, 509-518]. An improved structural preservation of the enzyme was observed in the presence of either of the activators acetyl-CoA (250 microM) and palmitoyl-CoA (1-5 microM). At higher than 5 microM palmitoyl-CoA, although activity was further increased, dissociation of enzyme tetramers was evident, presumably because of the detergent effect of the long-chain acyl moiety. Two inhibitors of yeast pyruvate carboxylase, L-aspartate (10 mM) and 2-oxoglutarate (40 mM), added alone or together decreased significantly the proportion of intact tetramers even in the presence of acetyl-CoA or palmitoyl-CoA. When yeast pyruvate carboxylase was incubated with avidin, the formation of unbranched linear concatemers occurred at avidin:enzyme ratios between 2:1 and 1:2. Avidin molecules were sometimes bound asymmetrically to the enzyme, appearing to complex only one biotin group on each side of the enzyme. This appeared to permit kinking and circularization of some concatemers.
D000495:Allosteric Site / Q000187:drug effects; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008958:Models, Molecular; D011485:Protein Binding; D011766:Pyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D046911:Macromolecular Substances; D001360:Avidin; D001710:Biotin; D011766:Pyruvate Carboxylase