The phosphate moiety of phosphoenolpyruvate does NOT contribute to allosteric regulation of liver pyruvate kinase by fructose-1,6-bisphosphate✝

ABSTRACTAllosteric regulation is central to the role of the glycolytic enzyme pyruvate kinase M2 (PKM2) in cellular metabolism. Multiple activating and inhibitory allosteric ligands regulate PKM2 activity by controlling the equilibrium between high activity tetramers and low activity dimers and monomers. However, it remains elusive how allosteric inputs upon simultaneous binding of different ligands are integrated to regulate PKM2 activity. Here, we show that, in the presence of the allosteric inhibitor L-phenylalanine (Phe), the activator fructose 1,6-bisphosphate (FBP) can induce PKM2 tetramerisation, but fails to maximally increase enzymatic activity. Guided by a new computational framework we developed to identify residues that mediate FBP-induced allostery, we generated two PKM2 mutants, A327S and C358A, in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings demonstrate a role for residues involved in FBP-induced allostery in enabling the integration of allosteric input from Phe and reveal a mechanism that underlies the co-ordinate regulation of PKM2 activity by multiple allosteric ligands.

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Allosteric Regulation of Human Liver Pyruvate Kinase by Peptides that Mimic the Phosphorylated/Dephosphorylated N-Terminus

Methods in Molecular Biology - Allostery ◽

10.1007/978-1-61779-334-9_18 ◽

2011 ◽

pp. 335-349 ◽

Cited By ~ 4

Author(s):

Charulata B. Prasannan ◽

Qingling Tang ◽

Aron W. Fenton

Keyword(s):

Pyruvate Kinase ◽

Human Liver ◽

Allosteric Regulation ◽

N Terminus

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Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate.

Journal of Bacteriology ◽

10.1128/jb.179.9.2987-2993.1997 ◽

1997 ◽

Vol 179 (9) ◽

pp. 2987-2993 ◽

Cited By ~ 82

Author(s):

E Boles ◽

F Schulte ◽

T Miosga ◽

K Freidel ◽

E Schlüter ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Pyruvate Kinase ◽

Fructose 1,6 Bisphosphate

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A study of the allosteric kinetics of Phycomyces pyruvate kinase as judged by the effect of l-alanine and fructose 1,6-bisphosphate

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology ◽

10.1016/0167-4838(86)90118-4 ◽

1986 ◽

Vol 874 (2) ◽

pp. 193-204 ◽

Cited By ~ 5

Author(s):

Pilar del Valle ◽

Dolores de Arriaga ◽

Felix Busto ◽

Joaquín Soler

Keyword(s):

Pyruvate Kinase ◽

Fructose 1,6 Bisphosphate ◽

Kinetics Of

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Partial purification and properties of pyruvate kinase and its regulatory role during lipid accumulation by the oleaginous yeast Rhodosporidium toruloides CBS 14

Canadian Journal of Microbiology ◽

10.1139/m85-089 ◽

1985 ◽

Vol 31 (5) ◽

pp. 479-484 ◽

Cited By ~ 11

Author(s):

Christopher Thomas Evans ◽

Colin Ratledge

Keyword(s):

Pyruvate Kinase ◽

Oleaginous Yeast ◽

Lipid Production ◽

Energy Charge ◽

Rhodosporidium Toruloides ◽

Regulatory Role ◽

Relative Mass ◽

Partial Purification ◽

Wide Range ◽

Fructose 1,6 Bisphosphate

Pyruvate kinase from the oleaginous yeast Rhodosporidium toruloides CBS 14 was partially purified and its properties investigated to determine its role during lipid production by this yeast. The enzyme (relative mass (Mr) = 190 000) showed a pH optimum of 8.0 and apparent Km values for K+, phosphoenolpyruvate (PEP), and ADP of 1.6 mM, 571 μM, and 120 μM, respectively. Enzyme activity was inhibited by citrate, isocitrate, ATP, GTP, and CTP and activated by fructose 1,6-bisphosphate, L-glutamate, and [Formula: see text] ions. Inhibition by citrate and ATP were both competitive with PEP with the Ki(citrate) = 340 μM and Ki(ATP) = 303 μM. The effect of ATP and cellular energy charge were critically dependent on the concentration of ADP present in the enzyme assay. Both L-glutamate and fructose 1,6-bisphosphate increased the affinity of the enzyme for both PEP and ADP and so were significant activators at nonsaturating substrate concentrations. [Formula: see text] ions increased the affinity of the enzyme for PEP, but not ADP. The modulation of pyruvate kinase activity by such a wide range of effectors is indicative of a major regulatory role in controlling the flux of carbon, through glycolysis, into lipid-synthesizing systems.

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