RESUMEN
Malic enzyme is a tetrameric protein with double dimer quaternary structure. In 3-5 M urea, the pigeon cytosolic NADP(+)-dependent malic enzyme unfolded and aggregated into various forms with dimers as the basic unit. Under the same denaturing conditions but in the presence of 4 mM Mn(2+), the enzyme existed exclusively as a molten globule dimer in solution. Similar to pigeon enzyme (Chang, G. G., T. M. Huang, and T. C. Chang. 1988. Biochem. J. 254:123-130), the human mitochondrial NAD(+)-dependent malic enzyme also underwent a reversible tetramer-dimer-monomer quaternary structural change in an acidic pH environment, which resulted in a molten globule state that is also prone to aggregate. The aggregation of pigeon enzyme was attributable to Trp-572 side chain. Mutation of Trp-572 to Phe, His, Ile, Ser, or Ala abolished the protective effect of the metal ions. The cytosolic malic enzyme was completely digested within 2 h by trypsin. In the presence of Mn(2+), a specific cutting site in the Lys-352-Gly-Arg-354 region was able to generate a unique polypeptide with M(r) of 37 kDa, and this polypeptide was resistant to further digestion. These results indicate that, during the catalytic process of malic enzyme, binding metal ion induces a conformational change within the enzyme from the open form to an intermediate form, which upon binding of L-malate, transforms further into a catalytically competent closed form.
Asunto(s)
Malato Deshidrogenasa/química , Malato Deshidrogenasa/ultraestructura , Metales/química , Estabilidad de Enzimas , Transición de Fase , Conformación Proteica , Pliegue de ProteínaRESUMEN
The chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH associated in several characteristic binding topologies either deep inside the cavity or at its inlet, contacting three to four consecutive GroEL apical domains. Consistent with visualization of bound polypeptide distributed over many parts of the central cavity, disulfide crosslinking could be carried out between a cysteine in a bound substrate protein and cysteines substituted anywhere inside GroEL. Finally, substrate binding induced adjustments in GroEL itself, observed mainly as clustering together of apical domains around sites of substrate binding.
Asunto(s)
Chaperonina 60/ultraestructura , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Pliegue de Proteína , Secuencias de Aminoácidos , Animales , Sitios de Unión , Chaperonina 60/química , Simulación por Computador , Microscopía por Crioelectrón/métodos , Cisteína/química , Disulfuros/química , Escherichia coli , Procesamiento de Imagen Asistido por Computador , Malato Deshidrogenasa/química , Malato Deshidrogenasa/ultraestructura , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , PorcinosRESUMEN
Intense synchrotron radiation produces specific structural and chemical damage to crystalline proteins even at 100 K. Carboxyl groups of acidic residues (Glu, Asp) losing their definition is one of the major effects observed. Here, the susceptibilities to X-ray damage of acidic residues in tetrameric malate dehydrogenase from Haloarcula marismortui are investigated. The marked excess of acidic residues in this halophilic enzyme makes it an ideal target to determine how specific damage to acidic residues is related to their structural and chemical environment. Four conclusions are drawn. (i) Acidic residues interacting with the side-chains of lysine and arginine residues are less affected by radiation damage than those interacting with serine, threonine and tyrosine side-chains. This suggests that residues with higher pK(a) values are more vulnerable to damage than those with a lower pK(a). However, such a correlation was not found when calculated pK(a) values were inspected. (ii) Acidic side-chains located in the enzymatic active site are the most radiation-sensitive ones. (iii) Acidic residues in the internal cavity formed by the four monomers and those involved in crystal contacts appear to be particularly susceptible. (iv) No correlation was found between radiation susceptibility and solvent accessibility.
Asunto(s)
Aminoácidos/química , Aminoácidos/efectos de la radiación , Cristalografía por Rayos X/métodos , Haloarcula marismortui/química , Malato Deshidrogenasa/química , Malato Deshidrogenasa/efectos de la radiación , Modelos Químicos , Simulación por Computador , Relación Dosis-Respuesta en la Radiación , Concentración de Iones de Hidrógeno , Malato Deshidrogenasa/ultraestructura , Modelos Moleculares , Conformación Proteica/efectos de la radiación , Desnaturalización Proteica/efectos de la radiación , Dosis de Radiación , Soluciones , Solventes/química , Relación Estructura-Actividad , Rayos XRESUMEN
For better understanding of the coenzyme specificity in NAD-dependent MDH (tMDH) from Thermus flavus AT-62, we determined the crystal structures of tMDH-NADP(H) complex at maximally 1.65 A resolution. The overall structure is almost the same as that of the tMDH-NADH complex. However, NADP(H) binds to tMDH in the reverse orientation, where adenine occupies the position near the catalytic center and nicotinamide is positioned at the adenine binding site of the tMDH-NADH complex. Consistent with this, kinetic analysis of the malate-oxidizing reaction revealed that NADP(+) inhibited tMDH at high concentrations. This has provided the first evidence for the alternative binding mode of the nicotinamide coenzyme, that has pseudo-symmetry in its structure, in a single enzyme.
Asunto(s)
Malato Deshidrogenasa/química , Malato Deshidrogenasa/ultraestructura , Modelos Químicos , Modelos Moleculares , NADP/química , NADP/ultraestructura , Sitios de Unión , Simulación por Computador , Cristalografía , Malato Deshidrogenasa/análisis , Malato-Deshidrogenasa (NADP+) , Complejos Multiproteicos/análisis , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , NADP/análisis , Unión Proteica , Conformación ProteicaRESUMEN
Human mitochondrial NAD(P)(+)-dependent malic enzyme was overexpressed in Escherichia coli and purified by anion-exchange, ATP affinity, and gel filtration chromatography. The protein was crystallized with the hanging-drop vapor diffusion method. Many different crystal forms were observed, five of which were characterized in some detail. A 2.5-A multiple-wavelength anomalous diffraction data set and a 2.1-A native data set were collected using synchrotron radiation on crystals containing selenomethionyl residues. These crystals belong to space group B2, with a = 204.4 A, b = 107.0 A, c = 59.2 A, and gamma = 101.9 degrees. Self-rotation functions demonstrated that the tetramer of this enzyme obeys 222 symmetry.
Asunto(s)
Malato Deshidrogenasa/química , Mitocondrias/química , Mitocondrias/enzimología , Cristalización , Cristalografía por Rayos X , Humanos , Malato Deshidrogenasa/ultraestructura , Selenometionina/químicaRESUMEN
The ontogeny of the cerebral pyruvate recycling pathway and the cellular localization of associated enzymes, malic enzyme (ME) and phosphoenolpyruvate carboxykinase (PEPCK), have been investigated using a combination of 13C NMR spectroscopy, enzymatic analysis, and molecular biology approaches. Activity of the pathway, using [1,2-(13)C2]acetate as a substrate, was detected by 13C NMR in brain extracts 3 weeks after birth, increasing progressively up to the third month of age. In whole-brain homogenates, ME activity increased to adult levels with the same time course as the recycling pathway. PEPCK activity was low during the first 2 weeks of life and decreased further toward adulthood. ME and PEPCK activity were found in primary cultures of astrocytes and in synaptosomal fractions of adult brain. Primary cultures of cortical neurons showed PEPCK activity but no detectable ME activity. The cytosolic ME gene was expressed in primary cultures of neurons and in astrocytes as well as in the neonatal and adult brain. The PEPCK gene was expressed both in primary cultures of cortical neurons and in astrocytes, but the level of its expression in the neonatal and adult brain was undetectable.
Asunto(s)
Encéfalo/metabolismo , Malato Deshidrogenasa/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Ácido Pirúvico/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/enzimología , Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Encéfalo/ultraestructura , Células Cultivadas , Espectroscopía de Resonancia Magnética , Malato Deshidrogenasa/biosíntesis , Malato Deshidrogenasa/ultraestructura , Neuronas/enzimología , Especificidad de Órganos , Fosfoenolpiruvato Carboxiquinasa (GTP)/biosíntesis , Fosfoenolpiruvato Carboxiquinasa (GTP)/ultraestructura , Ratas , Ratas Wistar , Sinaptosomas/enzimología , Células Tumorales CultivadasRESUMEN
Protein folding mediated by the molecular chaperone GroEL occurs by its binding to non-native polypeptide substrates and is driven by ATP hydrolysis. Both of these processes are influenced by the reversible association of the co-protein, GroES (refs 2-4). GroEL and other chaperonin 60 molecules are large, cylindrical oligomers consisting of two stacked heptameric rings of subunits; each ring forms a cage-like structure thought to bind polypeptides in a central cavity. Chaperonins play a passive role in folding by binding or sequestering folding proteins to prevent their aggregation, but they may also actively unfold substrate proteins trapped in misfolded forms, enabling them to assume productive folding conformations. Biochemical studies show that GroES improves the efficiency of GroEL function, but the structural basis for this is unknown. Here we report the first direct visualization, by cryo-electron microscopy, of a non-native protein substrate (malate dehydrogenase) bound to the mobile, outer domains at one end of GroEL. Addition of GroES to GroEL in the presence of ATP causes a dramatic hinge opening of about 60 degrees. GroES binds to the equivalent surface of the GroEL outer domains, but on the opposite end of the GroEL oligomer to the protein substrate.
Asunto(s)
Proteínas Bacterianas/ultraestructura , Proteínas de Choque Térmico/ultraestructura , Malato Deshidrogenasa/ultraestructura , Pliegue de Proteína , Adenosina Trifosfato/química , Animales , Proteínas Bacterianas/química , Chaperonina 10 , Chaperonina 60 , Escherichia coli , Congelación , Proteínas de Choque Térmico/química , Procesamiento de Imagen Asistido por Computador , Malato Deshidrogenasa/química , Unión Proteica , PorcinosRESUMEN
The structure of malate dehydrogenase from Escherichia coli complexed with the substrate analog, citrate and the cofactor NAD, has been determined by X-ray crystallography. A monoclinic crystal of the malate dehydrogenase, grown in citrate buffer, was soaked in 10 mM NAD solution and found to be isomorphous with the apo-form. The X-ray data extended to 1.9 A, nearly the same resolution limit as the apo-enzyme crystals. The ternary complex of malate dehydrogenase has very few conformational differences from that of the pseudo binary complex of enzyme with bound citrate. In addition, the NAD molecule has a very similar conformation to the NAD as found in the crystal structure of the cytosolic eukaryotic malate dehydrogenase. Similar hydrogen bond interactions are made by both enzymes from polar groups belonging to the NAD. Such interactions include hydrogen bonds from the ribose oxygens and the phosphate oxygens, to backbone amide and carbonyl atoms of the protein and to side-chains of a select few conserved hydrophilic residues. The only notable difference occurs in the active site region where the nicotinamide moiety is obstructed from further entering the active site by the C-6 carbonyl atoms of citrate. In this position there are no direct polar interactions between the protein and the nicotinamide moiety. Energy minimization of the structure with malate substituted for citrate in the active site shows that the nicotinamide moiety assumes the same position in the active site as the NAD in cytosolic malate dehydrogenase. The carboxamide atoms of the energy minimized model make significant hydrogen bond interactions with the catalytic residue, H177, and with the main-chain atoms of I117 and V146 in the vicinity of the active site, while the position of the rest of the cofactor remains unchanged.
Asunto(s)
Citratos/química , Malato Deshidrogenasa/ultraestructura , NAD/química , Sitios de Unión , Cristalografía , Escherichia coli/enzimología , Enlace de Hidrógeno , Difracción de Rayos XRESUMEN
The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3(1)21 or its enantiomer, with a = b = 131.2(7) A, c = 152.6(9) A, and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (lambda = 0.95 A) to about 3.0 A resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.