Human acylphosphatase cannot replace phosphoglycerate kinase in Saccharomyces cerevisiae.
Human acylphosphatase (h-AP, EC 3.6.1.7) has been reported to catalyse the hydrolysis of the 1-phosphate group of 1,3-diphosphoglycerate. In vivo operation of this reaction in the yeast Saccharomyces cerevisiae would bypass phosphoglycerate kinase and thus reduce the ATP yield from glycolysis. To investigate whether h-AP can indeed replace the S. cerevisiae phosphoglycerate kinase, a multi-copy plasmid carrying the h-AP gene under control of the yeast TDH3 promoter was introduced into a pgk1 delta mutant of S. cerevisiae. A strain carrying the expression vector without the h-AP cassette was used as a reference. For both strains, steady-state carbon- and energy-limited chemostat cultures were obtained at a dilution rate of 0.10 h(-1) on a medium containing a mixture of glucose and ethanol (15% and 85% on a carbon basis, respectively). Although the h-AP strain exhibited a high acylphosphatase activity in cell extracts, switching to glucose as sole carbon and energy source resulted in a complete arrest of glucose consumption and growth. The lack of a functional glycolytic pathway was further evident from the absence of ethanol formation in the presence of excess glucose in the culture. As h-AP cannot replace yeast phosphoglycerate kinase in vivo, the enzyme is not a useful tool to modify the ATP yield of glycolysis in S. cerevisiae.
Van Hoek
P
P
Kluyver Laboratory of Biotechnology, Delft University of Technology, The Netherlands.
Modesti
A
A
Ramponi
G
G
Kötter
P
P
van Dijken
J P
JP
Pron
J T
JT
D017766:Acid Anhydride Hydrolases / Q000235:genetics* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D003470:Culture Media; D006019:Glycolysis; D006801:Humans; D009154:Mutation; D010735:Phosphoglycerate Kinase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014170:Transformation, Genetic; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D000255:Adenosine Triphosphate; D003470:Culture Media; D010735:Phosphoglycerate Kinase; D000097024:Acylphosphatase