Kinetic properties of cerebral pyruvate kinase.
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ABSTRACT:
SUBMITTER: Nicholas PC
PROVIDER: S-EPMC1168063 | biostudies-other | 1974 Jul
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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Project description:Kinetic properties of rat liver pyruvate kinase type I at pH7.5 and 6.5 were studied with physiological ranges of substrates, modifiers and Mg(2+) concentrations at increasing enzyme concentrations, including the estimated cellular concentrations (approx. 0.1mg/ml). Enzyme properties appear unaffected by increased enzyme concentration if phosphoenolpyruvate, fructose 1,6-diphosphate and inhibitors are incubated with enzyme before starting the reaction with ADP. Our data suggest that minimum cellular concentrations of MgATP and l-alanine provide virtually complete inhibition of pyruvate kinase I at pH7.5. The most likely cellular control of existing pyruvate kinase I results from the strong restoration of enzyme activity by the small physiological amounts of fructose 1,6-diphosphate. Decreasing the pH to 6.5 also restores pyruvate kinase activity, but to only about one-third of its activity in the presence of fructose 1,6-diphosphate. Neither pyruvate nor 2-phosphoglycerate at cellular concentrations inhibit the enzyme significantly.
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Project description:Female Aedes aegypti mosquitoes are vectors of arboviruses that cause diseases of public health significance. The discovery of new metabolic targets is crucial for improving mosquito control strategies. We recently demonstrated that glucose oxidation supports ammonia detoxification in A. aegypti. Pyruvate kinase (PK, EC 2.7.1.40) catalyzes the last step of the glycolytic pathway. In most organisms, one or more allosteric effectors control PK activity. However, the kinetic properties and structure of PK in mosquitoes have not been previously reported. In this study, two alternatively spliced mRNA variants (AaPK1 and AaPK2) that code for PKs were identified in the A. aegypti genome. The AaPK1 mRNA variant, which encodes a 529 amino acid protein with an estimated molecular weight of ∼57 kDa, was cloned. The protein was expressed in Escherichia coli and purified. The AaPK1 kinetic properties were identified. The recombinant protein was also crystallized and its 3D structure determined. We found that alanine, glutamine, proline, serine and fructose-1-phosphate displayed a classic allosteric activation on AaPK1. Ribulose-5-phosphate acted as an allosteric inhibitor of AaPK1 but its inhibitory effect was reversed by alanine, glutamine, proline and serine. Additionally, the allosteric activation of AaPK1 by amino acids was weakened by fructose-1,6-bisphosphate, whereas the allosteric activation of AaPK1 by alanine and serine was diminished by glucose-6-phosphate. The AaPK1 structure shows the presence of fructose-1,6-bisphosphate in the allosteric site. Together, our results reveal that specific amino acids and phosphorylated sugars tightly regulate conformational dynamics and catalytic changes of AaPK1. The distinctive AaPK1 allosteric properties support a complex role for this enzyme within mosquito metabolism.
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Project description:The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 where the relationships between the initial velocities of the catalysed reaction and the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The findings were represented empirically by the exponential model for a regulatory enzyme. The analysis shows that ADP, phosphoenolpyruvate and Mg2+ display positive homotropic interaction in their binding behaviour with (calculated) Hill slopes at half-saturation equal to 1.06, 2.35 and 3.11 respectively [Ainsworth (1977) J. Theor. Biol. 68, 391-413]. The direct heterotropic interaction between ADP and phosphoenolpyruvate is small and negative, but the overall interaction between these substrates becomes positive when their positive interactions with Mg2+ are taken into account. The heterotropic interactions of the substrates, though smaller in magnitude, are comparable with those revealed by the rabbit muscle enzyme [Ainsworth, Kinderlerer & Gregory (1983) Biochem. J. 209, 401-411], and it is suggested that they have a common origin in charge interactions within the active site.
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