Summary
-
1.
The regulatory properties of two interconvertible kinetic forms of class A pyruvate kinase from Ehrlich ascites tumor cells have been studied with a partially purified enzyme preparation free of interfering enzymatic activities.
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2.
The hyperbolic form shows Michaelis-Menten kinetics for P-pyruvate, with high affinity for this substrate and low affinity for the inhibitory amino acids alanine and phenylalanine. The sigmoidal form displays positive cooperativity respect to P-pyruvate (n = 1.4), with lower affinity for this substrate and higher affinity for the inhibitory amino acids.
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3.
The equilibrium between the hyperbolic and the sigmoidal forms of the enzyme is affected by substrates and effectors. P-pyruvate, ADP and Fru-P2 shift the equilibrium to the hyperbolic form while ATP, alanine and phenylalanine stabilize the sigmoidal form.
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4.
Effector metabolites affect the molecular weight of the protein, acting on an equilibrium between dimers and tetramers. P-pyruvate and ADP associate the enzyme to a tetramer while ATP, alanine and phenylalanine favor the occurrence as a dimer. The positive modifier Fru-P2 did not associate the enzyme to the tetramer, even at 1mm concentration.
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5.
A tentative molecular model for pyruvate kinase A on the basis of the kinetic and aggregation interconversion is proposed.
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Abbreviations
- DTE:
-
dithioerythritol
- Fru-P2 :
-
fructose 1,6-bis-phosphate
- P-pyruvate:
-
phosphoenolpyruvate
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Felíu, J.E., Sols, A. Interconversion phenomena between two kinetic forms of class a pyruvate kinase from Ehrlich ascites tumor cells. Mol Cell Biochem 13, 31–44 (1976). https://doi.org/10.1007/BF01732393
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DOI: https://doi.org/10.1007/BF01732393