RESUMO
Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.
Assuntos
Glucose-6-Fosfato/química , Fosfoenolpiruvato Carboxilase/química , Fosfoenolpiruvato/química , Proteínas de Plantas/química , Zea mays/enzimologia , Regulação Alostérica , Domínio Catalítico , Glucose-6-Fosfato/metabolismo , Cinética , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Zea mays/genéticaRESUMO
Over-accumulation of triglycerides (TGs) in goose hepatocytes leads to the formation of fatty acid liver. Phosphoenolpyruvate carboxylase kinase 1 (PEPCK) is regarded as the rate-limiting enzyme for gluconeogenesis, and there is evidence that PEPCK is involved in regulating hepatic glucolipid metabolism. Hence, we proposed that PEPCK may have a role in goose hepatic steatosis. To test our hypothesis, the present study was conducted to firstly determine the sequence characteristics of goose PEPCK and then to explore its role in overfeeding-induced fatty liver. Our results showed that goose PEPCK encodes a 622-amino-acids protein that contains highly conserved oxaloacetate-binding domain, kinase-1 and kinase-2 motifs. PEPCK had higher mRNA levels in goose liver, and overfeeding markedly increased its expression in livers of both Sichuan White and Landes geese (p 0.05). Besides, expression of PEPCK was positively correlated with hepatic TG levels as well as plasma glucose and insulin concentrations. Additionally, in cultured goose primary hepatocyte, treatment with either oleic acid (0.8, 1.2 or 1.6 mM) or linoleic acid (0.125 or 0.25 mM) significantly (p 0.05) enhanced the expression of PEPCK. Taken together, these data suggested a role for PEPCK in the occurrence of overfeeding-induced goose hepatic steatosis.(AU)
Assuntos
Animais , Gansos/metabolismo , Gansos/fisiologia , Fosfoenolpiruvato Carboxilase/análise , Fosfoenolpiruvato Carboxilase/química , Fosfoenolpiruvato Carboxilase/genética , Fígado Gorduroso , HiperfagiaRESUMO
Over-accumulation of triglycerides (TGs) in goose hepatocytes leads to the formation of fatty acid liver. Phosphoenolpyruvate carboxylase kinase 1 (PEPCK) is regarded as the rate-limiting enzyme for gluconeogenesis, and there is evidence that PEPCK is involved in regulating hepatic glucolipid metabolism. Hence, we proposed that PEPCK may have a role in goose hepatic steatosis. To test our hypothesis, the present study was conducted to firstly determine the sequence characteristics of goose PEPCK and then to explore its role in overfeeding-induced fatty liver. Our results showed that goose PEPCK encodes a 622-amino-acids protein that contains highly conserved oxaloacetate-binding domain, kinase-1 and kinase-2 motifs. PEPCK had higher mRNA levels in goose liver, and overfeeding markedly increased its expression in livers of both Sichuan White and Landes geese (p 0.05). Besides, expression of PEPCK was positively correlated with hepatic TG levels as well as plasma glucose and insulin concentrations. Additionally, in cultured goose primary hepatocyte, treatment with either oleic acid (0.8, 1.2 or 1.6 mM) or linoleic acid (0.125 or 0.25 mM) significantly (p 0.05) enhanced the expression of PEPCK. Taken together, these data suggested a role for PEPCK in the occurrence of overfeeding-induced goose hepatic steatosis.
Assuntos
Animais , Fosfoenolpiruvato Carboxilase/análise , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/química , Fígado Gorduroso , Gansos/fisiologia , Gansos/metabolismo , HiperfagiaRESUMO
Two phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) isoforms of 74 and 65 kDa were found to coexist in vivo in pineapple leaves, a constitutive Crassulacean Acid Metabolism plant. The 65 kDa form was not the result of proteolytic cleavage of the larger form since extraction methods reported to prevent PEPCK proteolysis in other plant tissues failed to yield a single immunoreactive PEPCK polypeptide in leaf extracts. In this work, the smaller form of 65 kDa was purified to homogeneity and physically and kinetically characterized and showed parameters compatible with a fully active enzyme. The specific activity was nearly twice higher for decarboxylation of oxaloacetate when compared to carboxylation of phosphoenolpyruvate. Kinetic parameters fell within the range of those estimated for other plant PEPCKs. Its activity was affected by several metabolites, as shown by inhibition by 3-phosphoglycerate, citrate, malate, fructose-1,6-bisphosphate, l-asparagine and activation of the decarboxylating activity by succinate. A break in the Arrhenius plot at about 30°C indicates that PEPCK structure is responsive to changes in temperature. The results indicate that pineapple leaves contain two PEPCK forms. The biochemical characterization of the smaller isoform performed in this work suggests that it could participate in both carbon and nitrogen metabolism in vivo by acting as a decarboxylase.
Assuntos
Ananas/enzimologia , Fosfoenolpiruvato Carboxilase/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Descarboxilação , Ácido Oxaloacético/metabolismo , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxilase/química , Fotossíntese/fisiologia , Folhas de Planta/química , Proteínas de Plantas/química , Isoformas de Proteínas , TemperaturaRESUMO
The functional significance of tyrosine 207 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase was explored by examining the kinetic properties of the Tyr207Leu mutant. The variant enzyme retained the structural characteristics of the wild-type protein as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analyses of the mutated variant showed a 15-fold increase in K(m)CO2, a 32-fold decrease in V(max), and a 6-fold decrease in K(m) for phosphoenolpyruvate. These results suggest that the hydroxyl group of Tyr 207 may polarize CO2 and oxaloacetate, thus facilitating the carboxylation/decarboxylation steps.
Assuntos
Mutação/genética , Fosfoenolpiruvato Carboxilase/genética , Saccharomyces cerevisiae/enzimologia , Tirosina/genética , Catálise , Cromatografia em Gel , Dicroísmo Circular , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Fosfoenolpiruvato Carboxilase/química , Espectrometria de Fluorescência , Tirosina/químicaRESUMO
The functional signifcance of tyrosine 207 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase was explored by examining the kinetic properties of the Tyr207Leu mutant. The variant enzyme retained the structural characteristics of the wild-type protein as indicated by circular dichroism, intrinsic fuorescence spectroscopy, and gel-exclusion chromatography. Kinetic analyses of the mutated variant showed a 15-fold increase in Km CO2, a 32fold decrease in Vmax, and a 6-fold decrease in Km for phosphoenolpyruvate. These results suggest that the hydroxyl group of Tyr 207 may polarize CO2 and oxaloacetate, thus facilitating the carboxylation/decarboxylation steps.
Assuntos
Mutação/genética , Fosfoenolpiruvato Carboxilase/genética , Saccharomyces cerevisiae/enzimologia , Tirosina/genética , Catálise , Cromatografia em Gel , Dicroísmo Circular , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Fosfoenolpiruvato Carboxilase/química , Espectrometria de Fluorescência , Tirosina/químicaRESUMO
Spatial and temporal regulation of phosphoenolpyruvate carboxylase (PEPC) is critical to the function of C(4) photosynthesis. The photosynthetic isoform of PEPC in the cytosol of mesophyll cells in Kranz-type C(4) photosynthesis has distinctive kinetic and regulatory properties. Some species in the Chenopodiaceae family perform C(4) photosynthesis without Kranz anatomy by spatial separation of initial fixation of atmospheric CO(2) via PEPC from C(4) acid decarboxylation and CO(2) donation to Rubisco within individual chlorenchyma cells. We studied molecular and functional features of PEPC in two single-cell functioning C(4) species (Bienertia sinuspersici, Suaeda aralocaspica) as compared to Kranz type (Haloxylon persicum, Salsola richteri, Suaeda eltonica) and C(3) (Suaeda linifolia) chenopods. It was found that PEPC from both types of C(4) chenopods displays higher specific activity than that of the C(3) species and shows kinetic and regulatory characteristics similar to those of C(4) species in other families in that they are subject to light/dark regulation by phosphorylation and display differential malate sensitivity. Also, the deduced amino acid sequence from leaf cDNA indicates that the single-cell functioning C(4) species possesses a Kranz-type C(4) isoform with a Ser in the amino terminal. A phylogeny of PEPC shows that isoforms in the two single-cell functioning C(4) species are in a clade with the C(3) and Kranz C(4) Suaeda spp. with high sequence homology. Overall, this study indicates that B. sinuspersici and S. aralocaspica have a C(4)-type PEPC similar to that in Kranz C(4) plants, which likely is required for effective function of C(4) photosynthesis.
Assuntos
Chenopodiaceae/enzimologia , Evolução Molecular , Fosfoenolpiruvato Carboxilase/metabolismo , Fotossíntese/fisiologia , Sequência de Aminoácidos , Chenopodiaceae/genética , Ritmo Circadiano , Ponto Isoelétrico , Cinética , Dados de Sequência Molecular , Fosfoenolpiruvato Carboxilase/química , Fosfoenolpiruvato Carboxilase/genética , Fosforilação , Especificidade da EspécieRESUMO
At low concentrations of phosphoenolpyruvate and magnesium, the substrate of phosphoenolpyruvate carboxylase (PEPC) from Zea mays leaves is the MgPEP complex and free phosphoenolpyruvate (fPEP) is an allosteric activator [A. Tovar-Méndez, R. Rodríguez-Sotres, D.M. López-Valentín, R.A. Muñoz-Clares, Biochem. J. 332 (1998) 633-642]. To further the understanding of this photosynthetic enzyme, we have re-investigated its kinetics covering a 500-fold range in fPEP and free Mg(2+) (fMg(2+)) concentrations. Apparent V(max) values were dependent on the concentration of the fixed free species, suggesting that these species are substrates of the PEPC-catalyzed reaction. However, when substrate inhibition was taken into account, similar V(max) values were obtained in all saturation curves for a given varied free species, indicating that MgPEP is indeed the reaction substrate. As substrate inhibition may be the result of the rise in ionic strength of the assay medium, we studied its effects on the kinetics of the enzyme. Mixed inhibition against MgPEP was found, with apparent K(ic) and K(iu) values of 36 and 1370 mM, respectively. Initial velocity patterns determined at constant ionic strength, 600 mM, were consistent with MgPEP being the true PEPC substrate, fPEP an allosteric activator, and fMg(2+) a weak, non-competitive inhibitor, thus confirming the kinetic mechanism determined previously at low concentrations of PEP and Mg(2+), and indicating that apparent substrate inhibition by MgPEP in maize leaf PEPC is caused by inhibition by high magnesium and ionic strength.
Assuntos
Fosfoenolpiruvato Carboxilase/metabolismo , Zea mays/enzimologia , Cinética , Modelos Químicos , Concentração Osmolar , Fosfoenolpiruvato Carboxilase/química , Folhas de Planta/enzimologia , Especificidade por SubstratoRESUMO
The expression of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme (NADP-ME) in Egeria densa leaves was studied under low temperature and light (LTL) following incubation under high temperature and light (HTL), conditions previously shown to induce high and low CO(2) compensation points, respectively. Transfer from LTL to HTL conditions induced increases in the activities and amounts of both enzymes. One NADP-ME isoform was observed in induced and uninduced samples. Two isoforms of PEPC were expressed, with the lower M(r) isoform being induced by HTL. NADP-ME showed properties similar to those of the isoform in C(3) species. The inducible PEPC isoform has a low K(m) for both substrates. PEPC kinetic and regulatory properties (V(max) and K(m) for phosphoenolpyruvate, and I(50) for L-malate) are different in samples taken in the dark from those in the light, indicating that some modification of PEPC may be occurring during the day. Finally, abscisic acid induced the expression of PEPC and NADP-ME in a manner similar to temperature induction, except that the activities of both PEPC isoforms were increased. A different signaling system may exist in this species in response to high temperature or abscisic acid, both of which induce changes in photosynthetic metabolism.
Assuntos
Dióxido de Carbono/metabolismo , Magnoliopsida/metabolismo , Malato Desidrogenase/metabolismo , Fotossíntese/fisiologia , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Southern Blotting , Western Blotting , DNA de Plantas/análise , Isoenzimas/metabolismo , Luz , Magnoliopsida/enzimologia , Malatos/farmacologia , Peso Molecular , Fosfoenolpiruvato Carboxilase/química , Fosfoenolpiruvato Carboxilase/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , TemperaturaRESUMO
Incubation of the nonphosphorylated form of maize-leaf phospho enol pyruvate carboxylase (orthophosphate: oxaloacetate carboxy-lyase (phosphorylating), PEPC, EC 4.1.1.31) with the reagent pyridoxal 5'-phosphate (PLP) resulted in time-dependent, reversible inactivation and desensitization to the activator glucose 6-phosphate (Glc6P) and other related phosphorylated compounds. Both processes are not connected, since (i) when the PLP-modification was carried out in the presence of saturating ligands of the active site, which prevents inactivation, the desensitization to Glc6P is still observed, and (ii) under some experimental conditions the desensitization reaction is 4-times faster than the inactivation. Desensitization to Glc6P is first order with respect to PLP and has a second-order forward rate constant of 4.7 +/- 0.3 s-1 M-1 and a first-order reverse rate constant of 0.0046 +/- 0.0002 s-1. Correlation studies between the remaining Glc6P sensitivity and mol of PLP residues incorporated per mol of enzyme subunit indicate that one lysyl group for enzyme monomer is involved in the sensitivity of the enzyme to Glc6P. The reactivity of this group is increased by polyethylene glycol and glycerol, while the reactivity of the lysyl group of the active site is not affected by these organic cosolutes. In the presence but not in the absence of the organic cosolutes, Glc6P by itself offers significant protection against desensitization, while increases the extent of inactivation. Free PEP or PEP-Mg have opposite effects, protecting the enzyme against inactivation and increasing the degree of desensitization. They also increases the protection against desensitization afforded by Glc6P. Finally, the PEPC inhibitor malate provides some protection against both inactivation and desensitization. Taken together, these results are consistent with PLP-modification of a highly reactive lysyl group at or near the allosteric Glc6P-site.