Related Experiment Video
Updated: Aug 30, 2026

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
Published on: February 18, 2014
Mechanism of substrate inhibition in Escherichia coli phosphofructokinase
Aron W Fenton1, Gregory D Reinhart
1Department of Biochemistry and Biophysics and the Center for Advanced Biomolecular Research, Texas A&M University, 2128 TAMU, College Station, Texas 77843-2128, USA.
Abstract:
Phosphofructokinase from Escherichia coli (EcPFK) is a homotetramer with four active sites, which bind the substrates fructose-6-phosphate (Fru-6-P) and MgATP. In the presence of low concentrations of Fru-6-P, MgATP displays substrate inhibition. Previous proposals to explain this substrate inhibition have included both kinetic and allosteric mechanisms. We have isolated hybrid tetramers containing one wild type subunit and three mutated subunits (1:3). The mutated subunits contain mutations that decrease affinity for Fru-6-P (R243E) or MgATP (F76A/R77D/R82A) allowing us to systematically simplify the possible allosteric interactions between the two substrates. In the absence of a rate equation to explain the allosteric effects in a tetramer, the data have been compared to simulated data for an allosteric dimer. Since the apparent substrate inhibition caused by MgATP binding is not seen in hybrid tetramers with only a single native MgATP binding site, the proposed kinetic mechanism is not able to explain this phenomenon. The data presented are consistent with an allosteric antagonism between MgATP in one active site and Fru-6-P in a second active site.
Related Concept Videos
Enzymes
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
Feedback Inhibition
Enzyme Inhibition
Enzyme Kinetics
Scientists typically study enzyme kinetics with a fixed amount of enzyme in the controlled environment of a test tube. When more reactant, or substrate, is...
Introduction to Enzyme Kinetics
The experimenter can then plot the initial reaction rate or velocity (Vo) of a given trial against the substrate concentration ([S]) to obtain a graph of the reaction properties. For many enzymatic reactions involving a...
The Electron Transport Chain
Inhibitors of the electron transport chain
Rotenone, a widely used pesticide, prevents electron transfer from Fe-S cluster to ubiquinone or Q in...

