Some Catalytic and Regulatory Properties of Pyruvate Kinase from the Spadix and Retractor Muscles of Nautilus pompilius

Abstract

Pyruvate kinase was partially purified from the spadix and retractor muscles of Nautilus pompilius. In both cases, the enzyme was activated by magnesium and potassium ions with similar affinities (apparent Ka values were 0.63 ± 0.04 mM and 5.8 ± 0.4 mM, respectively, for the enzyme from the spadix; and 0.77 ± 0.06 mM and 6.7 ± 0.8 mM, respectively, for the enzyme from the retractor muscle). The enzymes showed normal hyperbolic saturation kinetics for the substrates adenosine Y-diphosphate and phosphoenolpyruvate, and the apparent Km values were identical when measured at saturating concentrations of the cosubstrate (apparent Km values were 0.28 ± 0.01 mM and 0.063 ± 0.005 mM, respectively, for the spadix). Adenosine 5'-triphosphate, alanine, and citrate were found to be inhibitors. The enzyme from the spadix was more susceptible to inhibition by alanine than that from the retractor muscle. For the latter enzyme, inhibition by alanine was noncompetitive with respect to phosphoenolpyruvate, but the inhibition was nonlinear; it also decreased the affinity for Mg2+. For the enzyme from the spadix, inhibition by alanine changed the saturation kinetics for phosphoenolpyruvate to sigmoidal form. The affinity for Mg2+ was also decreased by alanine. For both enzymes, fructose-I, 6-bisphosphate at a concentration of 0.05 mM partially reversed the inhibition by alanine, but not that by adenosine Y-triphosphate. The sigmoidal kinetics observed for phosphoenolpyruvate could also be reversed by increasing the concentration of Mg2 +. In general, the properties were found to be similar to those of other pyruvate kinases from the mantle muscle of squid and octopus, except for the observation of inhibition by alanine. These regulatory properties are discussed with respect to potential control of glycolytic flux during muscle activity.