Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate

Abstract

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.Pyruvate kinase is the last enzyme in the glycolytic pathway of sugar catabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate (PEP) into pyruvate by the addition of a proton and the loss of a phosphate group, which is transferred to ADP. Pyruvate kinases from a wide range of organisms have been extensively studied, and much is known about their physical and catalytic properties (8,35,38,39). Nearly all characterized eukaryotic pyruvate kinases are tightly regulated and are activated by fructose-1,6-bisphosphate (FBP). The mammalian muscle isoenzyme M1 is the only known pyruvate kinase that displays hyperbolic kinetics and lacks allosteric control (40). On the other hand, pyruvate kinases from prokaryotes can be activated by either FBP or other sugar phosphates (e.g., ribose phosphate), and those from trypanosomes can be activated by fructose-2,6-bisphosphate (35).In the yeast Saccharomyces cerevisiae, pyruvate kinase has been thought to be encoded solely by the PYK1 gene. The gene was cloned and sequenced by Burke et al. (16), and part of the nucleotide sequence was revised by McNally et al. (36). PYK1 codes for a 54.5-kDa protein (Pyk1p) consisting of 500 amino acids. Mutants defective in the PYK1 gene fail to grow on fermentable carbon sources and are even inhibited by them (17,18,30). However, they grow normally on ethanol or other gluconeogenic carbon sources. Under those conditions, hexose phosphates are provided by the gluconeogenic pathway, which uses the enzymes FBP and PEP carboxykinase to bypass the 6-phosphofructo-1-kinase and pyruvate kinase reactions, respectively. The concentrations of glycolytic metabolites in pyk1 deletion mutants after growth on an ethanol-containing medium are similar to the wild-type levels, but after addition of glucose, a large increase in the amounts of PEP and phosphoglycerates can be observed (12, 17). These results suggested that Pyk1p is the main enzyme which catalyzes the conversion of PEP into pyruvate in S. cerevisiae.The biochemical properties of yeast Pyk1p suggest that it plays a central regul...

Functioning of a metabolic flux sensor in Escherichia coli

“…We hope that future studies by us and others can provide additional support for the hypothesis put forward in this work. Notably, there is indication that flux-sensing mechanisms might also be present in other organisms: recently, it was reported that the budding yeast Saccharomyces cerevisiae shows a similar relationship between the glycolytic flux and FBP (7,38) and also its pyruvate kinase is feedforward activated by FBP (39). Thus, our findings are likely relevant also for other organisms.…”

In Vivo and in Vitro Phosphorylation of Two Isoforms of Yeast Pyruvate Kinase by Protein Kinase A

“…With the completion of the yeast genome sequencing project, an open reading frame coding for a protein with high homology to Pyk1 has been reported. The characterization of this gene indicated the existence of a Pyk2 protein, which is insensitive to FBP and whose expression is repressed by glucose (17). In this work we identify both Pyk1 and Pyk2 as in vitro and in vivo substrates of PKA, characterize their behavior as PKA substrates, and present a preliminary study of the effect of phosphorylation on the kinetics of pyruvate kinase.…”

“…The decrease in specific activity correlated with the levels of Pyk1 followed by Western blot. According to these results the kinetic experiments that follow were performed on partially purified preparations of Pyk from cells in a pre-diauxic shift stage, in which the specific activity of Pyk1 is maximal and there is no expression of Pyk2 (17).…”

Homeostasis and the glycogen shunt explains aerobic ethanol production in yeast

“…Yeast defective in this gene fail to grow on glucose, but they can grow on ethanol or other gluconeogenic carbon sources (37). Boles et al (37) discovered the existence of an isoform of PK, Pyk2p, which they reported "is subject to glucose repression and. .…”