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...

No abstract

We have cloned and characterized the two remaining unknown genes of the non-oxidative part of the pentose-phosphate pathway of Saccharomyces cerevisiae encoding the enzymes D-ribulose-5-phosphate 3-epimerase (Rpe1p) and D-ribose-5-phosphate ketol-isomerase (Rki1p). Rpe1p has an unexpected high specific activity of 2148 mU x (mg protein)-1 in crude extracts. Deletion mutants of RPE1 show no enzyme activity and are unable to grow on D-xylulose. Unexpectedly, haploid rki1 deletion mutants are not viable. Functional expression of RKI1 was demonstrated following an increase of gene dosage in the haploid rki1 deletion mutant, which restored viability and specific D-ribose-5-phosphate ketol-isomerase activity. Both enzymes show high similarity to the deduced protein sequences of various open reading frames, expressed sequence tags or cDNAs from different organisms.

We present a rapid, cheap and highly efficient method for site-directed mutagenesis using the polymerase chain reaction (PCR). This method is applicable to every DNA fragment which has to be cloned into the multiple cloning site of any vector, or vector pair, in two different orientations. It requires only two primers, one new and specific mutagenic primer and one of the usual sequencing primers. In the first PCR, a mutagenic DNA fragment is synthesized which is amplified exponentially in the second PCR. In contrast, wild-type sequences are only linearly amplified resulting in an efficiency of mutagenesis of nearly 100%.

Replacement of lysine144 by glutamine in the pentose phosphate pathway enzyme transaldolase of Saccharomyces cerevisiae is associated with the complete loss of activity indicating the essential role in catalysis. Neither histidine nor cysteine is important for catalytic activity as proposed for the Candida utilis enzyme. Also we could not find any evidence for a half-site character of the enzyme as described for transaldolase of C. utilis. Therefore, the reaction mechanisms for the two enzymes are different.