Phosphorylation of pyruvate kinase type K is restricted to the dimeric form.
In the absence of glycolytic intermediate, fructose-1,6-bisphosphate, pyruvate kinase type K exists in the dimeric form and is readily phosphorylated, whereas in the same sample and the same conditions pyruvate kinase type M is present as a tetramer and is not phosphorylated. Addition of fructose-1,6-bisphosphate results in the association of dimeric K2 molecules to a tetrameric K4 enzyme as determined by gel filtration and cellulose acetate electrophoresis, with concomitant loss of the capacity of the K isozyme to become phosphorylated. Phosphorylated K2 dimers can also tetramerize, but with a low recovery of the radiolabel, suggesting a fructose-1,6-bisphosphate induced dephosphorylation or selective degradation. The dimeric K isozyme is enzymatically active; inactive K-type monomers can be detected by immunoblot analysis in the absence of fructose-1,6-bisphosphate, but no phosphorylated pyruvate kinase is present in this fraction. The formation of K4 tetramers can not be accomplished by the substrate phosphoenolpyruvate. Fructose-1,6-bisphosphate is an allosteric activator of pyruvate kinase type K and induces hyperbolic saturation curves for phosphoenolpyruvate. In contrast, in the absence of effectors, pyruvate kinase type M exhibits Michaelis-Menten kinetics, but sigmoidal curves can be induced by the amino acid phenylalanine. However, even in the presence of phenylalanine, the M-type maintained its tetrameric configuration and did not serve as a substrate in the phosphorylation reaction. These findings argue for the importance of subunit interaction in the regulation of phosphorylation of pyruvate kinase.
Weernink
P A
PA
Department of Haematology, University Hospital, Utrecht, Netherlands.
Rijksen
G
G
Mascini
E M
EM
Staal
G E
GE
D001254:Astrocytoma / Q000201:enzymology*; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005635:Fructosediphosphates / Q000378:metabolism*; D005910:Glioma; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008875:Middle Aged; D009132:Muscles / Q000201:enzymology*; D010766:Phosphorylation; D011770:Pyruvate Kinase / Q000302:isolation & purification / Q000378:metabolism*; D012016:Reference Values; D013154:Spleen / Q000201:enzymology*
D005635:Fructosediphosphates; D007527:Isoenzymes; D046911:Macromolecular Substances; D011770:Pyruvate Kinase; C029063:fructose-1,6-diphosphate