Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: UNIPROT:P17174 (aspartate aminotransferase)
14,872 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using active enzyme centrifugation, it was demonstrated that pig heart cytoplasmic aspartate aminotransferase retains its dimeric conformation with a decrease in protein concentration down to 0.2 microgram/ml (50 mM L-aspartate, 5 mM alpha-ketoglutarate, 0.1 M NaCl, 0.05 M potassium-phosphate buffer, pH 7.4, 20 degrees C).
...
PMID:[Analysis of cytoplasmic aspartate aminotransferase using active enzyme centrifugation]. 674 8

Differential scanning calorimetry has been applied to study factors affecting the thermally induced denaturation of cytoplasmic aspartate aminotransferase, a dimeric pyridoxal enzyme. The consequences of binding of coenzyme and substrate derivatives to both the apo and holo forms of the enzyme were investigated and are interpreted in terms of the stabilization of the native form of the enzyme. The binding of pyridoxal phosphate coenzyme increases the thermal stability of the apoenzyme by approximately 27 kcal mol-1 as judged by the change in free energy differences between the native and denatured states of the protein. The stabilization produced by coenzyme binding to the apoprotein appears to be primarily due to the Schiff's base and phosphoryl moieties of the coenzyme; association of the pyridine ring component is without significant structural consequence. Pyridoxal phosphate binding to the subunits of the dimer occurs in a noncooperative fashion as judged by the appearance of transitions unique to the apo, holo, and intermediate enzyme forms in a calorimetric titration. Holoenzyme stability depends on the chemical nature of the catalytically significant group occupying the C-4' position of the bound coenzyme. The stabilization afforded by binding of the aldehyde form (pyridoxal phosphate) which exists as an internal Schiff's base with Lys 258 is diminished when this bond is chemically reduced or when the aldehyde is replaced by an amine (pyridoxamine phosphate). Apoenzyme is also shown to be stabilized by the presence of substrates in the absence of coenzyme. The differential scanning calorimetry results thus confirm previous findings derived from nuclear magnetic resonance studies on the ability of apoenzyme to bind substrates (Martinez-Carrion, M. Cheng, S., and Relimpio, A. (1973) J. Biol. Chem. 248, 2153-2160). Substrates and their analogues perturb the holoenzyme stability and the order of increasing influence on the pyridoxal form of the holoenzyme is aspartate, erythro-hydroxyaspartate, alpha-ketoglutarate, and alpha-methylaspartate. While all these compounds form stable binary enzyme-substrate complexes (Jenkins, W.T., and D'Ari, L. (1966) J. Biol. Chem. 541, 5667-5674), the complex with alpha-methylaspartate produces anomalous changes in the protein structure which are reflected in the calorimetric parameters. This suggests that caution be exercised in the use of analogues as substrate substitutes in crystallographic work. Differential scanning calorimetry also appears as a sensitive method with which to study the stereochemical dependence of ligand binding on enzyme-induced thermal stabilization. This is illustrated by the use of 4-carbon dicarboxylic acids where only those in the conformation favorable for binding are effective in stabilizing the holoenzyme.
...
PMID:Differential scanning calorimetry of cytoplasmic aspartate transaminase. 721 92

Native mitochondrial aspartate aminotransferase (AATase) is cleaved selectively by trypsin at the peptide bonds after Arg 26 or after Lys 31 yielding two shortened enzyme derivatives, AATase 27-410, and AATase 32-410. Recent x-ray crystallographic determination of the spatial structure of AATase has shown that the NH2-terminal segments of the two polypeptide chains of this dimeric enzyme pass in front of the active site clefts and form two separate junctions with the neighboring subunit which are not contiguous with the main subunit interface (Eichele, G., Ford, G. C., Glor, M., Jansonius, J. N., Mavrides, C., and Christen, P. (1979) J. Mol. Biol. 133, 161-180). The peptide bonds cleaved by trypsin are situated in the following stretch of the polypeptide chain which runs in exposed position on the surface of the subunit. The split-off peptide is lost during gel filtration. The molecular activity of AATase 27/32-410 (a mixture of about equal amounts of the two not readily separable derivatives) is about 3% of that of the native enzyme. In contrast, the K'm values for aspartate and 2-oxoglutarate are unchanged, indicating an unaltered geometry of the substrate binding site. A substantially diminished syncatalytic response of the reactivity of Cys 166 toward 5,5'-dithiobis-(2-nitrobenzoate) suggests that the decrease in catalytic activity is due to an interference with the syncatalytic conformational dynamics observed previously in AATase (Gehring, H., and Christen, P. (1978) J. Biol. Chem. 253, 3158-3163). Consonant with a role of the NH2-terminal segment in propagating the syncatalytic conformational rearrangements the rate of the tryptic cleavage is retarded 4-fold in the presence of the transaminating substrate pair aspartate and oxalacetate.
...
PMID:Mitochondrial aspartate aminotransferase 27/32-410. Partially active enzyme derivative produced by limited proteolytic cleavage of native enzyme. 743 Jan 25

The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC and 5'-methylthioadenosine, the committed step in ethylene biosynthesis in plants. Apple ACC synthase was overexpressed in Escherichia coli (3 mg/liter) and purified to near homogeneity. A continuous assay was developed by coupling the ACC synthase reaction to the deamination of 5'-methylthioadenosine by adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Aspergillus oryzae. The enzyme is dimeric, with kcat = 9s-1 per monomer and Km = 12 microM for AdoMet. The pyridoxal phosphate-binding site of ACC synthase appears to be highly homologous to that of aspartate aminotransferase, suggesting similar roles for corresponding residues. Site-directed mutagenesis of Lys-273, Arg-407, and Tyr-233 (corresponding to residues 258, 386, and 225 in aspartate aminotransferase) and kinetic analyses of the mutants confirms their importance in the ACC synthase mechanism. The Lys-273 to Ala mutant has no detectable activity, supporting the identification of this residue as the base catalyzing C alpha proton abstraction. Mutation of Arg-407 to Lys results in a precipitous drop in kcat/Km and an increase in Km for AdoMet of at least 20-fold, in accordance with its proposed role as principal ligand for the substrate alpha-carboxylate group. Replacement of Tyr-233 with Phe causes a 24-fold increase in the Km for AdoMet and no change in kcat, suggesting that this residue plays a role in orienting the pyridoxal phosphate cofactor in the active site.
...
PMID:Expression of apple 1-aminocyclopropane-1-carboxylate synthase in Escherichia coli: kinetic characterization of wild-type and active-site mutant forms. 780 54

We report here the x-ray studies of the complex cytosolic aspartate aminotransferase from chicken heart with D-aspartate at 2,7 A resolution. Crystals of the complex was prepared by diffusing D-aspartate into free enzyme crystals; their space group is P 2(1)2(1)2(1) with cell dimensions (A): a = 62.59; b = 117.83; c = 124.38. They contain one dimeric molecule in the asymmetric unit. The x-ray crystallographic analysis proves that the connection of the D-aspartate induces small conformational changes in the active site of two subunits of the enzyme: considerable conformational changes are determined for His 189, Phe 360, Tyr 70, Arg 292, Phe 18 and Glu 141.
...
PMID:[The complex of aspartate aminotransferase with D-aspartate]. 781 5

The three-dimensional structures of pyridoxamine 5'-phosphate-type aspartate aminotransferase from Escherichia coli and its complexes with maleate and glutarate have been determined by X-ray crystallography at 2.2, 2.1, and 2.7 A resolution, respectively. The enzyme is a dimeric form comprising two identical subunits, each of which is divided into one large and one small domain. The complex with maleate showed that substrate (or inhibitor) binding induced a large conformational change from the "open" to the "closed" form, resulting in closure of the active site by the small domain movement, as was observed in the pyridoxal 5'-phosphate-type enzyme. In the open form, three hydrophobic residues (hydrophobic plug) at the entrance of the active site are exposed to solvent. Maleate binding make the active site more hydrophobic by charge compensation and release of water molecules, facilitating the movement of the hydrophobic plug into the active site pocket to induce a large conformational change in the enzyme. Maleate is fixed rigidly in the active site pocket by extensive salt bridges and a hydrogen bonding network, guaranteeing the stereo-specificity of the catalysis and giving a Michaelis complex model. Contrary to our expectation, the glutarate complex was in the open form, suggesting that the equilibrium between the open and closed forms lies far toward the open form in solution. The water molecules located in the active site pocket were almost completely conserved between Escherichia coli and chicken mitochondrial aspartate aminotransferase with the same type of cofactor and the same conformation.
...
PMID:X-ray crystallographic study of pyridoxamine 5'-phosphate-type aspartate aminotransferases from Escherichia coli in three forms. 789 26

Following the chromatographic separation of the grey mullet (Mugil auratus Risso) red muscle extract, two fractions with aspartate aminotransferase activity were detected. One of the anticipated enzymes was purified to homogeneity. The isolated enzyme was a dimeric protein composed of identical subunits with the overall M(r) of about 65,000. It consisted of three electrophoretically distinct subforms with isoelectric points at pH 8.50, 8.70 and 8.85, respectively. The Michaelis-Menten constants of the substrates L-aspartate and 2-oxoglutarate were estimated to be 0.29 +/- 0.012 mM and 0.45 +/- 0.016 mM, respectively. For the reverse reaction, the Km for L-glutamate was 8.57 +/- 2.1 mM and for oxaloacetate it was 0.13 +/- 0.035 mM. The inhibition of the isolated enzyme by hydroxylamine was of a mixed linear noncompetitive type for L-aspartate as a substrate, whereas with 2-oxoglutarate hyperbolic uncompetitive inhibition was observed. The inhibition by aminooxyacetic acid and D,L-glyceraldehyde 3-phosphate was of a mixed linear noncompetitive type with respect to L-aspartate and 2-oxoglutarate. The isolated enzyme was slightly affected by maleate and succinate and no effects were produced by adipate. According to its subcellular distribution, susceptibility to inhibitors molecular and catalytic properties the isolated enzyme belonged to the mitochondrial form of aspartate aminotransferase.
...
PMID:Isolation and properties of mitochondrial aspartate aminotransferase from red muscle of grey mullet, Mugil auratus Risso. 791 42

The acid-induced reversible unfolding of several forms of the mitochondrial isoenzyme of mammalian aspartate aminotransferase, including its precursor form, has been characterized under equilibrium conditions. A minimum of two transitions can be detected for the holoenzyme (pyridoxal form). One transition takes place at pH 3.6 and corresponds to the monomerization of the dimeric protein. The second transition is centered at pH 3.3 and represents the disappearance of much of the tertiary and secondary structures. The presequence peptide in the precursor protein does not affect the equilibria nor the rate of unfolding in the pH range from 7.5 to 2.0. The presence of the cofactor, pyridoxal 5'-phosphate, stabilizes the protein against acid denaturation. At pH 2.0, the protein retains significant amounts of secondary structure (26% alpha-helix, 20% beta-structure). Increasing the ionic strength at pH 2.0 results in significant changes in the secondary structure of the unfolded protein that acquires some of the characteristics ascribed to a compact molten globule. According to the circular dichroism spectra these changes are characterized by an increase in beta-structure, although Fourier transform infrared spectroscopy analysis indicates that this increase in beta-structure is due mostly to the formation of intermolecular beta-sheet as a consequence of protein aggregation. The formation of high molecular weight aggregates has been confirmed by analytical ultracentrifugation. Following neutralization of the acid-unfolded state at low ionic strength both mature and precursor proteins refold to their native active state (> 80% yield). By contrast the compact state present at pH 2.0 and high ionic strength is unable to recover its activity following neutralization. Thus, this compact state does not appear to represent an intermediate in the folding pathway of the protein, but rather a dead end product of aggregation, which may reflect the intrinsic tendencies of the unfolded protein to oligomerize at intracellular salt concentrations unless controlled by factors such as chaperones present in the cellular environment.
...
PMID:Acid-induced reversible unfolding of mitochondrial aspartate aminotransferase. 807 19

The subunits of the alpha 2-dimeric enzyme aspartate aminotransferase are composed of two distinct domains, one large and one small. The active sites are situated close to both the intersubunit and the interdomain interface. Binding of substrate analogues to the active site induces a large conformational change in the enzyme, whereby the small domain rotates by 13 degrees relative to the large domain and completely buries the ligand. We have determined the crystal structures of chicken mitochondrial aspartate aminotransferase (mAATase) in two new crystal forms. A comparison of the structures of mAATase in five crystal forms, including both the unliganded and the liganded enzyme, shows that mAATase exists in either one of two unique conformations, with only minimal adaptations to the crystal lattice. This suggests that both the open, unliganded and closed, liganded structure of mAATase are, to a large extent, stabilized by intramolecular interactions, and are consequently representative of functional states of the protein in solution. A 2-fold-symmetric packing interaction between small domains occurring identically in three crystal forms of mAATase is described.
...
PMID:Crystalline mitochondrial aspartate aminotransferase exists in only two conformations. 812 Sep 3

We report here the first X-ray studies of the complex of cytosolic aspartate aminotransferase from chicken heart with D-aspartate at 2.5 A resolution. Crystals of the complex were grown by cocrystallization (space group is P2(1)2(1)2(1), parameters: a = 62.48 A, b = 117.71 A, c = 124.38 A). They contain one dimeric molecule in the asymmetric unit. The X-ray analysis proves that attachment of D-aspartate induces considerable conformational changes in the active sites of two subunits of the enzyme: both subunits of the complex are in the closed conformation, the interaction of the enzyme with D-aspartate induces a substantial turn (about 90 degrees) of the coenzyme in one subunit, the coenzyme ring is deformed, considerable conformational changes are determined for Phe-18 and Glu-141. Apparently, the amino group of the substrate is a trigger of the conformational changes in the active site of the enzyme.
...
PMID:[Study of crystals of aspartate aminotransferase complexed with D-aspartate]. 818 65


<< Previous 1 2 3 4 5 6 Next >>

---