Differential regulation of the yeast isozymes of pyruvate carboxylase and the locus of action of acetyl CoA.
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.
Jitrapakdee
Sarawut
S
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. scsji@mucc.mahidol.ac.th
Adina-Zada
Abdussalam
A
Besant
Paul G
PG
Surinya
Kathy H
KH
Cleland
W Wallace
WW
Wallace
John C
JC
Attwood
Paul V
PV
D000105:Acetyl Coenzyme A / Q000494:pharmacology*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000494:pharmacology; D001710:Biotin / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011766:Pyruvate Carboxylase / Q000235:genetics / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D007527:Isoenzymes; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D001710:Biotin; D000105:Acetyl Coenzyme A; D011766:Pyruvate Carboxylase