title: Reaction path of phosphofructo-1-kinase is altered by mutagenesis and alternative substrates.
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.
authors: Departments of Microbiology and Immunology and Biochemistry and Molecular Biology, The Chicago Medical School, North Chicago, Illinois 60064, USA.
authors: X
authors: X
authors: Wang
authors: R G
authors: RG
authors: Kemp
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000494:Allosteric Regulation / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005636:Fructosephosphates / Q000378:metabolism; D007700:Kinetics; D008745:Methylation; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D005636:Fructosephosphates; C032284:fructose-1-phosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; C068652:1-phosphofructokinase