Reaction Path of phosphofructo-1-kinase Is Altered by Mutagenesis and Alternative Substrates

Biochemistry. 2001 Apr 3;40(13):3938-42. doi: 10.1021/bi002709o.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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

Substances

  • Fructosephosphates
  • fructose-1-phosphate
  • adenosine 5'-O-(3-thiotriphosphate)
  • Adenosine Diphosphate
  • Phosphoenolpyruvate
  • Adenosine Triphosphate
  • Phosphofructokinase-1
  • 1-phosphofructokinase