PMID:11300773
| Citation |
Wang, X and Kemp, RG (2001) Reaction path of phosphofructo-1-kinase is altered by mutagenesis and alternative substrates. Biochemistry 40:3938-42 |
|---|---|
| Abstract |
Escherichia coli phosphofructokinase (PFK) has been proposed to have a random, nonrapid equilibrium mechanism that produces nonallosteric ATP inhibition as a result of substrate antagonism. The consequences of such a mechanism have been investigated by employing alternative substrates and mutants of the enzyme that produce a variety of nonallosteric kinetic patterns demonstrating substrate inhibition and sigmoid velocity curves. Mutations of a methionine residue in the sugar phosphate binding site produced apparent cooperativity in the interaction of fructose 6-phosphate. Cooperativity could also be seen with native enzyme using a poorly binding substrate, fructose 1-phosphate. With an alternative nucleotide, 1-carboxymethyl-ATP, coupled with a mutation that introduced a negative charge in the nucleotide binding site, one could observe substrate inhibition by fructose 6-phosphate and apparent cooperativity in the interaction with nucleotide. Furthermore, the use of a phosphoryl donor, gamma-thiol-ATP, which greatly reduced the catalytic rate, apparently facilitated the equilibration of all binding reactions and eliminated ATP inhibition. These unusual kinetic patterns could be interpreted within the random, steady-state model as reflecting changes in the rates of particular binding and catalytic events. |
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| Keywords |
Adenosine Diphosphate/metabolism; Adenosine Triphosphate/analogs & derivatives; Adenosine Triphosphate/metabolism; Allosteric Regulation/genetics; Amino Acid Substitution/genetics; Binding Sites/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Fructosephosphates/metabolism; Kinetics; Methylation; Models, Chemical; Mutagenesis, Site-Directed; Phosphoenolpyruvate/metabolism; Phosphofructokinase-1/antagonists & inhibitors; Phosphofructokinase-1/genetics; Phosphofructokinase-1/metabolism; Phosphorylation; Substrate Specificity/genetics |
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