The role of Glu187 in the regulation of phosphofructokinase by phosphoenolpyruvate.
In bacterial phosphofructokinases, either a glutamic or an aspartic residue is present at position 187, and the mechanism of inhibition by phosphoenolpyruvate seems to be correlated to the nature of residue 187. Upon binding phosphoenolpyruvate, only the enzymes with a Glu187 would undergo a major allosteric conformational change from an active into an inactive state, whereas the enzymes with an Asp187 would only show a simple upward shift in their pH-profile of activity. The phosphofructokinase from Spiroplasma citri, which has an Asp187, has been purified and its properties follow this pattern. The behaviour of mutants of the enzyme from Escherichia coli in which Glu187 is replaced by either aspartate or leucine confirms the importance of residue 187. The major allosteric transition of E. coli phosphofructokinase is abolished by the substitution Glu187-->Asp, suggesting that a glutamate at position 187 is necessary (but not sufficient) for the protein to undergo the change from the active into the inactive state induced by phosphenolpyruvate. In addition, the presence of an acidic residue, aspartate or glutamate, at position 187 is required (but not sufficient) for the binding of ADP (or GDP). This requirement of a negative charge for ADP binding could explain the striking conservation of an aspartate residue at position 187 in all the eukaryotic phosphofructokinases.
Auzat
I
I
Laboratoire d'Enzymologie du CNRS, Gif-sur-Yvette, France.
Le Bras
G
G
Branny
P
P
De La Torre
F
F
Theunissen
B
B
Garel
J R
JR
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D005971:Glutamates; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010728:Phosphoenolpyruvate / Q000378:metabolism* / Q000494:pharmacology; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013149:Spiroplasma / Q000201:enzymology*
D005971:Glutamates; D018698:Glutamic Acid; D010728:Phosphoenolpyruvate; D010732:Phosphofructokinase-1