Catalytic and Regulatory Properties of Pyruvate Kinases in Tissues of a Marine Bivalve

Abstract

Abstract Pyruvate kinase (EC 2.7.1.40) from oyster tissues occurs in at least two electrophoretically and kinetically distinguishable forms in mantle tissue and in adductor muscle. In mantle, pyruvate kinase activity appears as a single band having a pI value of 6.35 while adductor pyruvate kinase appears as a doublet having pI values of 5.6 and 6.5. Both forms of the enzyme have an essential requirement for Mg++ and a monovalent cation (K+ or NH4+). The activation of oyster enzymes was more efficient with K+. Neither of the enzymes is inhibited by Cu++. In the absence of fructose 1,6-diphosphate, the Km(adp) for adductor muscle pyruvate kinase is several-fold higher than the Km(adp) for the mantle enzyme. In the presence of fructose-1,6-P2, the Km(adp) for both enzymes are identical. The Km of the substrate phosphoenolpyruvate differs for the two enzymes, Km(pep) being several-fold lower for the adductor enzyme. In the absence of fructose-1,6-P2, the Km(pep) for both enzymes decreases markedly with increasing pH; the Km(pep) is strikingly decreased by FDP and becomes insensitive to pH in presence of fructose-1,6-P2. For oyster pyruvate kinases fructose-1,6-P2 stimulation is maximal at acidic pH. Both forms of the enzymes are subject to feedback inhibition by ATP, l-alanine, and phenylalanine, and these inhibitory effects are reversed by fructose-1,6-P2. Ki values of these metabolites for adductor pyruvate kinase are lower than the Ki values of mantle enzyme. ATP inhibition differs for the two enzymes, being noncompetitive for the mantle form and competitive for the adductor enzyme. l-Alanine inhibition of both enzymes is of the mixed competitive type. Phenylalanine inhibition is competitive with respect to P-enolpyruvate for the mantle enzyme, but not for the adductor pyruvate kinase. The data in this study suggest that, as in the mammalian case, the enzyme pyruvate kinase in the oyster occurs in tissue specific multimolecular forms and that the kinetic properties of each isozyme seem to gear in well with the over-all metabolism of the tissue.