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The regulatory properties of yeast pyruvate kinase. Effects of NH4+ and K+ concentrations.

ABSTRACT: The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied at 25 degrees C and pH 6.2 as a function of the concentrations of ADP, phosphoenolpyruvate, Mg2+ and either NH4+ or K+. The data were analysed by the exponential model for four substrates, obtained by extension of the model described by Ainsworth, Kinderlerer & Gregory [(1983) Biochem. J. 209, 401-411]. On that basis, it was concluded that NH4+ binding is almost non-interactive but leads to the appearance of positive interaction in the velocity response to increase in its concentration because of positive interactions with phosphoenolpyruvate and Mg2+. The data obtained with K+ lead to the same conclusions and differ only in suggesting that NH4+ is bound more strongly to the enzyme than is K+. Both data sets are used as the basis for a discussion of the substrate interactions of pyruvate kinase and it appears therefrom that the heterotropic interactions accord with what is known of the events that take place at the active site during catalysis. The paper also reports a determination of the dissociation constants for the NH4+ complexes with ADP and phosphoenolpyruvate and an examination of the simultaneous activation of pyruvate kinase by K+ and NH4+ ions.

SUBMITTER: Rhodes N

PROVIDER: S-EPMC1146629 | biostudies-other | 1986 Mar

REPOSITORIES: biostudies-other

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Project description:The kinetic and regulatory properties of cytosolic pyruvate kinase (PKc) isolated from endosperm of germinating castor oil seeds (Ricinus communis L.) have been studied. Optimal efficiency in substrate utilization (in terms of Vmax/Km for phosphoenolpyruvate or ADP) occurred between pH 6.7 and 7.4. Enzyme activity was absolutely dependent on the presence of a bivalent and a univalent metal cation, with Mg2+ and K+ fulfilling this requirement. Mg2+ binding showed positive and negative co-operativity at pH 6.5 (h = 1.6) and pH 7.2 (h = 0.69) respectively. Hyperbolic saturation kinetics were observed with phosphoenolpyruvate (PEP) and K+, whereas ADP acted as a mixed-type inhibitor over 1 mM. Glycerol (10%, v/v) increased the S0.5(ADP) 2.3-fold and altered the pattern of nucleotide binding from hyperbolic (h = 1.0) to sigmoidal (h = 1.79) without modifying PEP saturation kinetics. No activators were identified. ATP, AMP, isocitrate, 2-oxoglutarate, malate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-phosphoglycerate, glycerol 3-phosphate and phosphoglycolate were the most effective inhibitors. These metabolites yielded additive inhibition when tested in pairs. ATP and 3-phosphoglycerate were mixed-type inhibitors with respect to PEP, whereas competitive inhibition was observed for other inhibitors. Inhibition by malate, 2-oxoglutarate, phosphorylated triose sugars or phosphoglycolate was far more pronounced at pH 7.2 than at pH 6.5. Although 32P-labelling studies revealed that extensive phosphorylation in vivo of soluble endosperm proteins occurred between days 3 and 5 of seed germination, no alteration in the 32P-labelling pattern of 5-day-germinated endosperm was observed after 30 min of anaerobiosis. Moreover, no evidence was obtained that PKc was a phosphoprotein in aerobic or anoxic endosperms. It is proposed that endosperm PKc activity of germinating castor seeds is enhanced after anaerobiosis through concerted decreases in ATP levels, cytosolic pH and concentrations of several key inhibitors.

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The regulatory properties of yeast pyruvate kinase. Effects of NH4+ and K+ concentrations.

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