Mutations in phosphofructokinases alter the control characteristics of glycolysis in vivo in Saccharomyces cerevisiae

Abstract

Ethanol and CO2 production from glucose by non-proliferating suspensions of aerobically-grown, glucose-derepressed wild-type Saccharomyces cerevisiae is inhibited by O2; monitoring by mass spectrometry provides a direct method for measurement of the Pasteur effect. Under aerobic conditions, that part of the CO2 evolved equivalent to the O2 consumed, is produced by respiration: subtraction of this respiratory CO2 from the total gives CO2 produced by aerobic glycolysis. Pasteur quotients (anaerobic CO2/aerobic glycolytic CO2) were within the range 1.2 to 3.0. The Pasteur effect was not observed in the presence of carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of mitochondrial energy metabolism, or in a rho degree cytoplasmic petite mutant. A 'non-allosteric' mutant with an altered regulatory subunit of phosphofructokinase showed no Pasteur effect. Strains bearing a nonsense mutation pfk1 in the catalytic subunit of soluble phosphofructokinase (PFKI) also showed no Pasteur effect; the residual fermentative activity of this strain was dependent on PFKII, the particulate phosphofructokinase. A double mutant lacking both PFKI and glucose-6-phosphate dehydrogenase showed similar characteristics to those of the single pfk1 mutant; this indicates that the hexose monophosphate shunt is not acting to bypass the phosphofructokinase block. A 'hyper-allosteric' mutant altered in the regulatory subunit encoded by the gene PFK2 showed characteristics of glucose fermentation and ethanol oxidation very similar to those of wild-type organisms. These results indicate that either of the two phosphofructokinases can carry out glycolysis.

Serum homocysteine, folate, vitamin B12 levels and arterial stiffness in diabetic patients: which of them is really important in atherogenesis?

“…Phosphofructokinase is the limiting enzyme of glycolysis, acting as a key regulator to this metabolic pathway (Lloyd et al , 1992; Arvanitidis and Heinisch, 1994; Reuter et al , 2000). The conventional cdc − phenotype and the abnormal septation pattern observed in cells overexpressing a cDNA coding for Pfk1p (see Figure 5a,b), suggest that this protein may play a dual function in glycolysis and cell cycle regulation that could serve to coordinate this main metabolic pathway with progression throughout the cell cycle.…”

The importance of ATP as a regulator of glycolytic flux inSaccharomyces cerevisiae

“…However, a number of key reactions have been identified, such as glucose transport and phosphorylation, phosphofructokinase and pyruvate kinase activity (Blazquez et al ., 1993; Campbell‐Burk and Shulman, 1987; Cortassa and Aon, 1997; Ernandes et al , 1998; Gancedo and Serrano, 1989; Müller et al ., 1997; Oehlen et al ., 1994). Especially, phosphofructokinase is a well‐studied enzyme (Davies and Brindle, 1992; Gancedo and Serrano, 1989; Lloyd et al ., 1992; Müller et al ., 1997; Reibstein et al ., 1986) and for a long time this enzyme was even considered to be the (single) rate‐limiting step of glycolysis. However, the development of metabolic control analysis theory has shown this to be an oversimplification (Fell, 1997).…”

Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

“…9), whereas Maitra and co-workers use the reverse designations, as in Ref. 10). Deletion mutants in either one of the genes still ferment glucose and are capable of growth on this carbon source, whereas pfk1 pfk2 double deletions are glucose-negative (11).…”