Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate.
We have characterized the gene YOR347c of Saccharomyces cerevisiae and shown that it encodes a second functional pyruvate kinase isoenzyme, Pyk2p. Overexpression of the YOR347c/PYK2 gene on a multicopy vector restored growth on glucose of a yeast pyruvate kinase 1 (pyk1) mutant strain and could completely substitute for the PYK1-encoded enzymatic activity. PYK2 gene expression is subject to glucose repression. A pyk2 deletion mutant had no obvious growth phenotypes under various conditions, but the growth defects of a pyk1 pyk2 double-deletion strain were even more pronounced than those of a pyk1 single-mutation strain. Pyk2p is active without fructose-1,6-bisphosphate. However, overexpression of PYK2 during growth on ethanol did not cause any of the deleterious effects expected from a futile cycling between pyruvate and phosphoenolpyruvate. The results indicate that the PYK2-encoded pyruvate kinase may be used under conditions of very low glycolytic flux.
Boles
E
E
Institut für Mikrobiologie, Heinrich-Heine-Universität, Düsseldorf, Germany. boles@uni-duesseldorf.de
Schulte
F
F
Miosga
T
T
Freidel
K
K
Schlüter
E
E
Zimmermann
F K
FK
Hollenberg
C P
CP
Heinisch
J J
JJ
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D000431:Ethanol / Q000378:metabolism; D005635:Fructosediphosphates / Q000378:metabolism*; D017353:Gene Deletion; D005800:Genes, Fungal; D005838:Genotype; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D009838:Oligodeoxyribonucleotides; D011770:Pyruvate Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism
D005635:Fructosediphosphates; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D000431:Ethanol; D011770:Pyruvate Kinase; D001616:beta-Galactosidase; D005947:Glucose; C029063:fructose-1,6-diphosphate