Purification and characterization of pyruvate kinase from Schizosaccharomyces pombe: evidence for an unusual quaternary structure.
Earlier attempts to purify and characterize nonrecombinant pyruvate kinase from Schizosaccharomyces pombe proved difficult due to problems associated with the instability of the protein. The enzyme has been overexpressed in Saccharomyces cerevisiae strain AH22, permitting studies to determine the conditions required to stabilize the enzyme during purification. Recombinant S. pombe pyruvate kinase was purified by a combination of ion-exchange chromatography and gel filtration. The purified enzyme showed sigmoidal kinetics with respect to PEP; in the presence of FBP, the kinetics were restored to Michaelis-Menten behavior. With respect to ADP, the Hill coefficient was not affected by FBP. Determination of the molecular mass of the purified enzyme by ultracentrifugation showed that it behaved as a dimer-tetramer system with a Kd of approximately 1 microM.
Nairn
J
J
Department of Biological and Molecular Sciences, University of Stirling, Stirling, FK9 4LA, Scotland, United Kingdom.
Duncan
D
D
Gray
L M
LM
Urquhart
G
G
Binnie
M
M
Byron
O
O
Fothergill-Gilmore
L A
LA
Price
N C
NC
D000244:Adenosine Diphosphate / Q000378:metabolism; D005635:Fructosediphosphates / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry; D015870:Gene Expression / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012568:Schizosaccharomyces / Q000201:enzymology*; D014461:Ultracentrifugation
D005635:Fructosediphosphates; D005656:Fungal Proteins; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; C029063:fructose-1,6-diphosphate