Partial purification and preliminary characterization of pyruvate kinase from the brown algaAscophyllum nodosum

The enzyme pyruvate kinase (EC 2.7.1.40) was partially purified from the marine brown alga Ascophyllum nodosum by means of DEAE-cellulose anion-exchange chromatography. The pH optimum for activity of this enzyme was 7·0 and the activation energy 62·2 kJ mol-1. In common with pyruvate kinase from other plants, the enzyme had a requirement for both K+ and Mg2+ (K a 2·46 and 1·15 mm, respectively). Substrate kinetics for ADP were hyperbolic (K m 0·15 mm), but those for phosphoenolpyruvate were sigmoid with a distinct positive cooperativity (K 0.5 1·68 mm, Hill coefficient 2·05). Fructose-6-phosphate (10 mm) acted as an allosteric activator, restoring Michaelis-Menten type kinetics for phosphoenolpyruvate (Hill coefficient 1·12) and substantially decreasing the K m (0·259 mm). Ascophyllum nodosum pyruvate kinase was also activated by AMP, and inhibited by ATP, CaCl2, citrate and oxalate.