The mechanism of ATP inhibition of wild type and mutant phosphofructo-1-kinase from Escherichia coli.
Escherichia coli 6-phosphofructo-1-kinase was inhibited by high concentrations of ATP at alkaline pH. The mechanism of the inhibition was studied with two mutants generated by site-directed mutagenesis; I126A, with a Km for fructose-6-P that was more than two orders of magnitude higher than that of wild type but with minimal changes in kcat and Km for ATP, and R72H, with little change in substrate half-saturation concentrations but with a kcat that was 300-fold lower that of wild type enzyme. ATP and fructose-6-P interacted in a mutually antagonistic manner; that is ATP decreased the apparent affinity for fructose-6-P and vice versa. The half-saturation concentrations for both substrates, most strikingly fructose-6-P, increased with increasing pH while the kcat increased. Studies with I126A suggested that ATP inhibition was not dependent on a dissociable group with a pK in the alkaline range and that the inhibition was not caused by abortive binding of substrate to the wrong substrate site. Inhibition was not the result of differential affinity of ATP for the R and T states of the enzyme. The low kcat mutant, R72H, did not display ATP inhibition. These data indicate that ATP inhibition results from substrate antagonism coupled with a steady state random mechanism wherein the high rate of catalysis does not permit equilibration of substrates.
Zheng
R L
RL
Department of Biological Chemistry, University of Health Sciences, Chicago Medical School, Illinois 60064.
Kemp
R G
RG
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005636:Fructosephosphates / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification
D005636:Fructosephosphates; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; C068652:1-phosphofructokinase