Pyruvate kinases from flight muscle and fat body of the cockroach,Periplaneta americana, were purified to homogeneity. The two tissues contained different forms of the enzyme which were separable by starch gel electrophoresis and isoelectric focusing (pI=5.75 for flight muscle and 6.15 for fat body). Both enzymes had molecular weights of 235,000±20,000.
Flight muscle pyruvate kinase displayed Michaelis-Menten kinetics with respect to both ADP and P-enolpyruvate withKm values of 0.27 and 0.04 mM, respectively.Km for Mg2+ was 0.60 mM andKa for K+ was 15 mM. The enzyme was weakly inhibitied by four compounds, ATP, arginine-P,l-alanine and citrate with apparentKi values of 3.5, 15, 20 and 24 mM, respectively. Competitive inhibition by 3 mM ATP or 10 mM arginine-P raised theKm for P-enolpyruvate to 0.067 or 0.057 mM. Fructose-1,6-P2 did not activate the enzyme but reversed inhibitions by ATP and arginine-P.
Fat body pyruvate kinase showed sigmoidal kinetics with respect to P-enolpyruvate with S0.5=0.32 mM andnH=1.43.Km values for ADP and Mg2+ were 0.30 and 0.80 mM, respectively with aKa for K+ of 10 mM. ATP andl-alanine were inhibitors of the enzyme; 2 mM ATP raised S0.5 for P-enolpyruvate to 0.48 mM while 3 mMl-alanine increased S0.5 to 0.84 mM. Neither citrate nor arginine-P inhibited the enzyme but citrate affected the enzyme by reversingl-alanine inhibition. Fat body pyruvate kinase was strongly activated by fructose-1,6-P2 with an apparentKa of 1.5 M. Fructose-1,6-P2 at 0.1 mM reduced S0.5 for P-enolpyruvate to 0.05 mM andnH to 1.0.
Flight muscle and fat body pyruvate kinases from the cockroach show properties analogous to those of the muscle and liver forms of mammalian pyruvate kinase. Fat body pyruvate kinase is suited for on-off function in a tissue with a gluconeogenic capacity. Strong allosteric control with a feed-forward activation by fructose-1,6-P2 is key to coordinating enzyme function with glycolytic rate. The function of flight muscle pyruvate kinase in energy production during flight is aided by a lowKm for P-enolpyruvate, weak inhibitor effects by high energy phosphates and deinhibition of these effects by fructose-1,6-P2.