ALLOSTERIC PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE .2. KINETICS OF NATIVE AND THIOL-MODIFIED ENZYME

The kinetics of native skeletal muscle phosphofructokinase were compared with the kinetic properties of phosphofructokinase that had one thiol group modified per protomer by reaction with 5,5′-dithiobis(2-nitrobenzoic acid). Native phosphofructokinase displayed the following properties. (1) As the concentration of adenosine 5′-triphosphate was increased in the assay, the Hill interaction coefficient and the concentration of fructose 6-phosphate necessary to achieve half-maximal velocity also increased. No cooperative interactions were observed with inosine 5′-triphosphate as the phosphoryl donor. (2) At pH 7.35, the interaction coefficient and concentration of fructose 6-phosphate necessary for half-maximal velocity were much lower than that observed at pH 7.0. (3) In the presence of adenosine 5-monophosphate or inorganic phosphate, both the interaction coefficient and the half-maximal velocity concentration of fructose 6-phosphate were decreased. The following results were obtained with studies of the thiol-modified enzyme. (1) Maximum activity was decreased by about two-thirds. (2) The pH-activity profile was unchanged by the modification. (3) Modification had no influence on the interaction of fructose 6-phosphate or nucleoside triphosphate with the catalytic site on the enzyme as determined with inosine 5′-triphosphate as the phosphoryl donor. (4) With adenosine 5′-triphosphate as the phosphoryl donor, modification greatly reduced the observed interaction coefficient but did not significantly reduce the half-maximal velocity concentration of fructose 6-phosphate. As a result, the modification activates the enzyme under the assay conditions of high concentration of adenosine 5′-triphosphate and low concentrations of fructose 6-phosphate. The kinetics of native and thiol-modified phosphofructokinase are discussed in relation to a model that proposes two conformational states for the enzyme, and this model is compared with present views of allosteric enzymes. It is proposed that thiol modification does not restrict interaction of magnesium adenosine 5′-triphosphate with the inhibitory site but instead limits the complete conformational expression of that interaction. © 1969, American Chemical Society. All rights reserved.