Regulatory properties of hexokinase from flight muscle of Schistocerca americana gregaria. Role of the enzyme in control of glycolysis during the rest-to-flight transition

Hexokinase (E.C.2.7.1.1) from the flight muscle of the locust, Schistorcerca americana gregaria, was purified to homogeneity by an affinity chromatography method. Hexokinase in locust muscle is a soluble enzyme, occurs as a single isozyme, and has a pI of 5.7, a molecular weight of 97,000 ± 4000, and a pH optimum of 7.6 in Tris buffer. Michaelis constants are 0.40 ± 0.04, 1.00 ± 0.11, and 0.15 ± 0.02 mM for ATP, Mg2+, and glucose respectively. ITP, CTP, GTP and UTP were poor phosphate donors for the enzyme but mannose and fructose could substitute for glucose and Mn2+ could satisfy the divalent cation requirement. High concentrations of K+, NH4 +, Li+, SO4 2- and Ca2+ (Ki(Ca2+) = 8.5±0.80 mM) were inhibitory. Product inhibition by ADP was non-competitive (Ki = 3.5 ± 0.3 mM) with respect to either substrate while product inhibition by glucose-6-P was non-competitive with respect to glucose (Ki = 0.087 ± 0.01 mM) and competitive with respect to ATP (Ki = 0.012 ± 0.001 mM). Pi, alanine and sn-glycerol-3-P could reverse glucose-6-P inhibition. All three altered the Vmax activity for glucose-6-P inhibited hexokinase while alanine also lowered the apparent Km for ATP. Alanine effects were potentiated by physiological levels of Na+. The regulation of flight muscle hexokinase in vivo during the rest-to-flight transition is discussed