Mutations in phosphofructokinases alter the control characteristics of glycolysis in vivo in Saccharomyces cerevisiae.
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.
Lloyd
D
D
Microbiology Group (PABIO), University of Wales College of Cardiff, U.K.
James
C J
CJ
Maitra
P K
PK
D000332:Aerobiosis; D000693:Anaerobiosis; D002245:Carbon Dioxide / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004252:DNA Mutational Analysis; D005285:Fermentation; D006019:Glycolysis; D006600:Hexosephosphates / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics; D007535:Isomerases / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D010101:Oxygen Consumption; D010732:Phosphofructokinase-1 / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D006600:Hexosephosphates; D007527:Isoenzymes; D002245:Carbon Dioxide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D010732:Phosphofructokinase-1; D007535:Isomerases