Search tips
Search criteria 


Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. 1997 May; 179(9): 2987–2993.
PMCID: PMC179064

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.

Full Text

The Full Text of this article is available as a PDF (330K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • al-Habori M. Microcompartmentation, metabolic channelling and carbohydrate metabolism. Int J Biochem Cell Biol. 1995 Feb;27(2):123–132. [PubMed]
  • Barwell CJ, Hess B. Application of kinetics of yeast pyruvate kinase in vitro to calculation of glycolytic flux in the anaerobic yeast cell. Hoppe Seylers Z Physiol Chem. 1972 Jul;353(7):1178–1184. [PubMed]
  • Barwell CJ, Woodward B, Brunt RV. Regulation of pyruvate kinase by fructose 1,6-diphosphate in Saccharomyces cerevisiae. Eur J Biochem. 1971 Jan 1;18(1):59–64. [PubMed]
  • Bennetzen JL, Hall BD. Codon selection in yeast. J Biol Chem. 1982 Mar 25;257(6):3026–3031. [PubMed]
  • Berben G, Dumont J, Gilliquet V, Bolle PA, Hilger F. The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast. 1991 Jul;7(5):475–477. [PubMed]
  • Boles E, Heinisch J, Zimmermann FK. Different signals control the activation of glycolysis in the yeast Saccharomyces cerevisiae. Yeast. 1993 Jul;9(7):761–770. [PubMed]
  • Boles E, Lehnert W, Zimmermann FK. The role of the NAD-dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant. Eur J Biochem. 1993 Oct 1;217(1):469–477. [PubMed]
  • Boles E, Zimmermann FK. Induction of pyruvate decarboxylase in glycolysis mutants of Saccharomyces cerevisiae correlates with the concentrations of three-carbon glycolytic metabolites. Arch Microbiol. 1993;160(4):324–328. [PubMed]
  • Boles E, Zimmermann FK. Open reading frames in the antisense strands of genes coding for glycolytic enzymes in Saccharomyces cerevisiae. Mol Gen Genet. 1994 May 25;243(4):363–368. [PubMed]
  • Brewster NK, Val DL, Walker ME, Wallace JC. Regulation of pyruvate carboxylase isozyme (PYC1, PYC2) gene expression in Saccharomyces cerevisiae during fermentative and nonfermentative growth. Arch Biochem Biophys. 1994 May 15;311(1):62–71. [PubMed]
  • Burke RL, Tekamp-Olson P, Najarian R. The isolation, characterization, and sequence of the pyruvate kinase gene of Saccharomyces cerevisiae. J Biol Chem. 1983 Feb 25;258(4):2193–2201. [PubMed]
  • Ciriacy M, Breitenbach I. Physiological effects of seven different blocks in glycolysis in Saccharomyces cerevisiae. J Bacteriol. 1979 Jul;139(1):152–160. [PMC free article] [PubMed]
  • Clifton D, Weinstock SB, Fraenkel DG. Glycolysis mutants in Saccharomyces cerevisiae. Genetics. 1978 Jan;88(1):1–11. [PubMed]
  • Dohmen RJ, Strasser AW, Höner CB, Hollenberg CP. An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast. 1991 Oct;7(7):691–692. [PubMed]
  • Eberhardt I, Hohmann S. Strategy for deletion of complete open reading frames in Saccharomyces cerevisiae. Curr Genet. 1995 Mar;27(4):306–308. [PubMed]
  • Entian KD. Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast. Mol Gen Genet. 1980;178(3):633–637. [PubMed]
  • Entian KD, Barnett JA. Regulation of sugar utilization by Saccharomyces cerevisiae. Trends Biochem Sci. 1992 Dec;17(12):506–510. [PubMed]
  • Gancedo JM. Carbon catabolite repression in yeast. Eur J Biochem. 1992 Jun 1;206(2):297–313. [PubMed]
  • Gietz RD, Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. [PubMed]
  • Hedges D, Proft M, Entian KD. CAT8, a new zinc cluster-encoding gene necessary for derepression of gluconeogenic enzymes in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1995 Apr;15(4):1915–1922. [PMC free article] [PubMed]
  • Heinisch JJ, Boles E, Timpel C. A yeast phosphofructokinase insensitive to the allosteric activator fructose 2,6-bisphosphate. Glycolysis/metabolic regulation/allosteric control. J Biol Chem. 1996 Jul 5;271(27):15928–15933. [PubMed]
  • Heinisch JJ, Valdés E, Alvarez J, Rodicio R. Molecular genetics of ICL2, encoding a non-functional isocitrate lyase in Saccharomyces cerevisiae. Yeast. 1996 Oct;12(13):1285–1295. [PubMed]
  • Hess B, Haeckel R. Interaction between potassium-, ammonium- and fructose-1,6-diphosphate activation of yeast pyruvate kinase. Nature. 1967 May 20;214(5090):848–849. [PubMed]
  • Kawasaki G, Fraenkel DG. Cloning of yeast glycolysis genes by complementation. Biochem Biophys Res Commun. 1982 Oct 15;108(3):1107–1122. [PubMed]
  • Ma H, Bloom LM, Walsh CT, Botstein D. The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Dec;9(12):5643–5649. [PMC free article] [PubMed]
  • Maitra PK, Lobo Z. A kinetic study of glycolytic enzyme synthesis in yeast. J Biol Chem. 1971 Jan 25;246(2):475–488. [PubMed]
  • Maitra PK, Lobo Z. Yeast pyruvate kinase: a mutant from catalytically insensitive to fructose 1,6-bisphosphate. Eur J Biochem. 1977 Sep;78(2):353–360. [PubMed]
  • Mattevi A, Bolognesi M, Valentini G. The allosteric regulation of pyruvate kinase. FEBS Lett. 1996 Jun 24;389(1):15–19. [PubMed]
  • McNally T, Purvis IJ, Fothergill-Gilmore LA, Brown AJ. The yeast pyruvate kinase gene does not contain a string of non-preferred codons: revised nucleotide sequence. FEBS Lett. 1989 Apr 24;247(2):312–316. [PubMed]
  • Muirhead H. Isoenzymes of pyruvate kinase. Biochem Soc Trans. 1990 Apr;18(2):193–196. [PubMed]
  • Muirhead H, Clayden DA, Barford D, Lorimer CG, Fothergill-Gilmore LA, Schiltz E, Schmitt W. The structure of cat muscle pyruvate kinase. EMBO J. 1986 Mar;5(3):475–481. [PubMed]
  • Myers AM, Tzagoloff A, Kinney DM, Lusty CJ. Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. Gene. 1986;45(3):299–310. [PubMed]
  • Navas MA, Gancedo JM. The regulatory characteristics of yeast fructose-1,6-bisphosphatase confer only a small selective advantage. J Bacteriol. 1996 Apr;178(7):1809–1812. [PMC free article] [PubMed]
  • Noguchi T, Inoue H, Tanaka T. The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. J Biol Chem. 1986 Oct 15;261(29):13807–13812. [PubMed]
  • Purnelle B, Goffeau A. Nucleotide sequence analysis of a 40 kb segment on the right arm of yeast chromosome XV reveals 18 open reading frames including a new pyruvate kinase and three homologues to chromosome I genes. Yeast. 1996 Nov;12(14):1475–1481. [PubMed]
  • Ronne H. Glucose repression in fungi. Trends Genet. 1995 Jan;11(1):12–17. [PubMed]
  • Rose M, Albig W, Entian KD. Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII. Eur J Biochem. 1991 Aug 1;199(3):511–518. [PubMed]
  • Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. [PubMed]
  • Srere PA. Complexes of sequential metabolic enzymes. Annu Rev Biochem. 1987;56:89–124. [PubMed]
  • Stucka R, Dequin S, Salmon JM, Gancedo C. DNA sequences in chromosomes II and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains. Mol Gen Genet. 1991 Oct;229(2):307–315. [PubMed]
  • Tu J, Carlson M. REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae. EMBO J. 1995 Dec 1;14(23):5939–5946. [PubMed]
  • Valdés-Hevia MD, de la Guerra R, Gancedo C. Isolation and characterization of the gene encoding phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae. FEBS Lett. 1989 Dec 4;258(2):313–316. [PubMed]
  • Winston F, Dollard C, Ricupero-Hovasse SL. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast. 1995 Jan;11(1):53–55. [PubMed]
  • Yin Z, Smith RJ, Brown AJ. Multiple signalling pathways trigger the exquisite sensitivity of yeast gluconeogenic mRNAs to glucose. Mol Microbiol. 1996 May;20(4):751–764. [PubMed]
  • Yun SL, Aust AE, Suelter CH. A revised preparation of yeast (Saccharomyces cerevisiae) pyruvate kinase. J Biol Chem. 1976 Jan 10;251(1):124–128. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)