Mutant studies of phosphofructo-2-kinases do not reveal an essential role of fructose-2, 6-bisphosphate in the regulation of carbon fluxes in yeast cells

Institut for Mikrobiologie und Genetik, Technische Hochschule Darmstadt, Schnittspahnstr. 10, D-64287 Darmstadt, Germany 2 Author for correspondence: Eckhard Boles. Tel: +49 211 81 12778. Fax: +49 211 81 15370. e-mail: boles@uni-duesseldorf.de ABSTRACT Summary: The effect of the allosteric regulator fructose-2, 6-bisphosphate (F2, 6bP) on the regulation of carbohydrate metabolism was investigated in vivo with Saccharomyces cerevisiae mutants containing no, very high or unregulated 6-phosphofructo-2-kinase activity. Simultaneous overproduction of F2, 6bP and 6-phosphofructo-1-kinase activity did not increase the glycolytic flux to ethanol. Overexpression of fructose-1, 6-bisphosphatase during growth on glucose in a mutant strain devoid of F2, 6bP did not cause pronounced effects on the cells. Moreover, high levels of F2, 6bP during growth on ethanol in a strain with a highly active 6-phosphofructo-2-kinase enzyme did not affect either carbon flux to glycogen or growth rate. Site-directed mutagenesis of 6-phosphofructo-2-kinase (Pfk26) revealed that serine 644 is involved in the activation of Pfk26 by protein kinase A phosphorylation, but that, additionally, the enzyme can be further activated by phosphorylation of another amino acid residue. The results demonstrate that F2, 6bP is not needed to sustain an adequate glycolytic flux under fermentative conditions, but rather is concerned with the homeostasis of metabolite concentrations. Moreover, they fail to indicate a physiological significance for inhibition of fructose-1,6-bisphosphatase by F2,6bP. Keywords: Saccharomyces cerevisiae , fructose-2,6-bisphosphate, 6-phosphofructo-1-kinase, glycolytic flux, futile cycling Present address: Institut für Mikrobiologie, Universität Düsseldorf, Universitatsstr. 1, Geb. 26.12.01, D-40225 Düsseldorf, Germany