Differential regulation of the yeast isozymes of pyruvate carboxylase and the locus of action of acetyl CoA

Int J Biochem Cell Biol. 2007;39(6):1211-23. doi: 10.1016/j.biocel.2007.03.016. Epub 2007 Mar 30.

Abstract

Unlike other eukaryotes studied to date, yeast has two genes for pyruvate carboxylase coding for very similar, but not identical, isozymes (Pyc1 and Pyc2), both of which are located in the cytoplasm. We have found that there are marked differences in the kinetic properties of the isozymes potentially leading to differential regulation of Pyc1 and Pyc2 activity by both activators and substrates. For example, Pyc2 is only activated 3.7-fold by acetyl CoA, and 9.6-fold by NH(4)(+), whilst the figures for Pyc1 are 16 and 14.6-fold, respectively. Pyc1 and Pyc2 display different allosteric properties with respect to acetyl CoA activation and aspartate inhibition, with Pyc1 showing a higher degree of cooperativity than Pyc2, even in the absence of aspartate. We have investigated the locus of action in the amino acid sequence of the isozymes of this activator by measuring its regulation of various chimeric constructs of the two isozymes. In this way, we conclude that the main locus of action of acetyl CoA lies in the N-terminal half of the enzyme, within the biotin-carboxylation domain, between amino acids 99 and 478 of Pyc1.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyl Coenzyme A / pharmacology*
  • Amino Acid Sequence
  • Aspartic Acid / pharmacology
  • Biotin / metabolism
  • Enzyme Activation / drug effects
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Pyruvate Carboxylase / genetics
  • Pyruvate Carboxylase / metabolism*
  • Quaternary Ammonium Compounds / pharmacology
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Homology, Amino Acid

Substances

  • Isoenzymes
  • Quaternary Ammonium Compounds
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Aspartic Acid
  • Biotin
  • Acetyl Coenzyme A
  • Pyruvate Carboxylase