Regulatory properties of pyruvate kinase from liver of the summer-active arctic ground squirrel.

Abstract

The properties of liver pyruvate kinase (PyK) from summer-active Arctic ground squirrel (Citellus undulatus) were examined over the physiological temperature range of the animal. One form of the enzyme with a pI (isoelectric point) value of 5.3 was observed and exhibited kinetics similar to those of L-type PyK. This form has a molecular weight of 243 000, similar to that of M-type PyK. Enzyme–phosphoenolpyruvate (PEP) affinity falls with decreasing temperature while affinity for ADP increases under these conditions. The effect of temperature upon extent of negative cooperativity of PEP binding and its possible physiological importance are briefly discussed. Fructose-1,6-phosphate (FDP) increases the affinity of PyK for PEP but has no effect upon enzyme-ADP interaction. However, Vmax in the presence of saturating concentrations of both substrates is increased two to threefold by FDP, the allosteric activator. Ground squirrel PyK is inhibited by ATP. Inhibition by ATP of PyK activity is diminished by decreasing the temperature or by increasing the concentrations of substrate. FDP reverses this inhibition but the degree of reversal is sensitive to substrate concentration and temperature. Inhibition by ATP is competitive for the PEP site and appears to be of the "mixed-competitive" variety when carried out at varying concentrations of ADP. Inhibition by alanine is noncompetitive for both substrate sites. As a result of the very high concentrations of amino acids needed to obtain appreciable inhibition of live PyK, this does not appear to be a physiological means of regulation of enzyme activity. The effect of intermediates of the glycolytic, pentose shunt, and Krebs acid cycle pathways upon PyK activity is briefly discussed. Inosine nucleotides, involved in the reversal of the PyK reaction by phosphoenolpyruvate carboxykinase, have a strong modulating effect upon PyK activity. These data suggest formal mechanisms for the regulation of liver PyK in the ground squirrel.