Identification of C-terminal motifs responsible for transmission of inhibition by ATP of mammalian phosphofructokinase, and their contribution to other allosteric effects.
Systematic deletions and point mutations in the C-terminal extension of mammalian PFK (phosphofructokinase) led us to identify Leu-767 and Glu-768 of the M-type isoform (PFK-M) as the motifs responsible for the role of this region in inhibition by MgATP. These amino acids are the only residues of the C-terminus that are conserved in all mammalian isoforms, and were found to have a similar function in the C-type isoenzyme. Both residues in PFK-C and Leu-767 in PFK-M were also observed to be critical for inhibition by citrate, which is synergistic with that by MgATP. Binding studies utilizing titration of intrinsic protein fluorescence indicated that the C-terminal part of the enzyme participates in the signal transduction route from the MgATP inhibitory site to the catalytic site, but does not contribute to the binding of this inhibitor, whereas it is essential for the binding of citrate. Mutations of the identified structural motifs did not alter either the action of other allosteric effectors that also interact with MgATP, such as the inhibitor phosphoenolpyruvate and the strong activator fructose 2,6-bisphosphate, or the co-operative effect of fructose 6-phosphate. The latter data provide evidence that activation by fructose 2,6-bisphosphate and fructose 6-phosphate co-operativity are not linked to the same allosteric transition as that mediating inhibition by MgATP.
Martínez-Costa
Oscar H
OH
Departamento de Bioquímica de la UAM and Instituto de Investigaciones Biomédicas Alberto Sols UAM-CSIC, Facultad de Medicina de la Universidad Autónoma de Madrid, 28029 Madrid, Spain.
Hermida
Carmen
C
Sánchez-Martínez
Cristina
C
Santamaría
Belén
B
Aragón
Juan J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019343:Citric Acid / Q000378:metabolism / Q000494:pharmacology; D006639:Histidine / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008322:Mammals / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D025221:Phosphofructokinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D007527:Isoenzymes; D019343:Citric Acid; D006639:Histidine; D000255:Adenosine Triphosphate; D025221:Phosphofructokinases