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Enzymes of Energy Metabolism in a Vertebrate Facultative Anaerobe, Pseudemys scripta

1974; Elsevier BV; Volume: 249; Issue: 5 Linguagem: Inglês

10.1016/s0021-9258(19)42898-6

1083-351X

Kenneth B. Storey, Peter W. Hochachka,

Erythrocyte Function and Pathophysiology

Abstract Pyruvate kinase (EC 2.7.1.40), extracted from turtle heart and purified to a specific activity of 210 µmoles of product per min per mg of protein, displays an isoelectric point of 6.05; its divalent cation requirements can be satisfied with Mg2+ or Mn2+, the Michaelis constant for Mg2+ being about 5-fold greater than for Mn2+. K+ or NH4+ can satisfy the requirement for a monovalent cation. Unlike the mammalian enzyme, the turtle heart pyruvate kinase shows an acid pH optimum (pH 6.3 to 6.5), and this property is considered of functional significance during sustained anoxia when intracellular pH drops significantly. Michaelis constants for P-enolpyruvate and ADP are 0.117 and 0.30 mm, respectively, and saturation of the enzyme with either subsubstrate follows hyperbolic kinetics. GDP and IDP can substitute for ADP as phosphate acceptors, both exhibiting sigmoidal saturation kinetics. The maximum velocities obtained with GDP and IDP are about 25% of that observed with ADP. In contrast to the mammalian muscle enzyme, the turtle enzyme is under tight metabolite regulation. It is potently inhibited by ATP, alanine, and phenylalanine, the respective Ki values being 3.7, 0.09, and 0.35 mm. ATP inhibition is noncompetitive with respect to ADP and is mixed competitive with respect to P-enolpyruvate. Alanine and phenylalanine inhibitions are both competitive with respect to P-enolpyruvate. Fructose diphosphate not only reverses, but overrides, all inhibitory effects of ATP, alanine, and phenylalanine. Moreover, fructose diphosphate directly activates turtle heart pyruvate kinase by a large increase in the apparent enzyme-substrate affinity. These effects of fructose diphosphate, coupled with fructose diphosphate control of turtle heart phosphofructokinase (Storey, K. B., and Hochachka, P. W. (1974) J. Biol. Chem. 249, 1417– 1422), suggest a particularly pivotal role for this metabolite in glycolytic control of turtle myocardium.