UNIPROT:P17174 - FACTA Search

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

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

A simple method was established for determination of the stereospecificity of C-4' hydrogen transfer of the coenzymes (pyridoxal and pyridoxamine). The method is based on the findings that aspartate aminotransferase of pig heart and D-amino acid aminotransferase of Bacillus sp. YM-1 catalyze the abstraction of the pro-S and pro-R proton at C-4' of pyridoxamine, respectively. Pyridoxal is a poor coenzyme, but readily released from the enzyme. It reacts in 3H2O with a substrate amino acid and an apo-aminotransferase whose stereospecificity for C-4' hydrogen transfer is to be determined. The resultant pyridoxamine which is tritiated at C-4' is incubated with an apo form of aspartate aminotransferase or D-amino acid aminotransferase and a substrate, alpha-keto acid. The stereospecificity for the C-4' hydrogen transfer examined is determined by measurement of radioactivity retained in the pyridoxal formed. We showed by means of this method that C-4' hydrogen transfer of coenzyme occurs on the si face of the external Schiff base in the transamination reactions of two aspartate aminotransferases of Bacillus sp. YM-2 and Escherichia coli, and aromatic amino acid aminotransferase of E. coli.
...
PMID:A simple method for determination of stereospecificity of aminotransferases for C-4' hydrogen transfer of the coenzyme. 785 65

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

The crystal structures of the stable, closed complexes of chicken mitochondrial aspartate aminotransferase with the natural substrates L-aspartate and L-glutamate have been solved and refined at 2.4- and 2.3-A resolution, respectively. In both cases, clear electron density at the substrate-coenzyme binding site unequivocally indicates the presence of a covalent intermediate. The crystallographically identical environments of the two subunits of the alpha 2 dimer allow a simple, direct correlation of the coenzyme absorption spectra of the crystalline enzyme with the diffraction results. Deconvolution of the spectra of the crystalline complexes using lognormal curves indicates that the ketimine intermediates constitute 76% and 83% of the total enzyme populations with L-aspartate and L-glutamate, respectively. The electron density maps accommodate the ketimine structures best in agreement with the independent spectral data. Crystalline enzyme has a much higher affinity for keto acid substrates compared to enzyme in solution. The increased affinity is interpreted in terms of a perturbation of the open/closed conformational equilibrium by the crystal lattice, with the closed form having greater affinity for substrate. The crystal lattice contacts provide energy required for domain closure normally supplied by the excess binding energy of the substrate. In solution, enzyme saturated with amino/keto acid substrate pairs has a greater total fraction of intermediates in the aldehyde oxidation state compared to crystalline enzyme. Assuming the only difference between the solution and crystalline enzymes is in conformational freedom, this difference suggests that one or more substantially populated, aldehydic intermediates in solution exist in the open conformation. Quantitative analyses of the spectra indicate that the value of the equilibrium constant for the open-closed conformational transition of the liganded, aldehydic enzyme in solution is near 1. The C4' pro-S proton in the ketimine models is oriented nearly perpendicularly to the plane of the pyridine ring, suggesting that the enzyme facilitates its removal by maximizing sigma-pi orbital overlap. The absence of a localized water molecule near Lys258 dictates that ketimine hydrolysis occurs via a transiently bound water molecule or from an alternative, possibly more open, structure in which water is appropriately bound. A prominent mechanistic role for flexibility of the Lys258 side chain is suggested by the absence of hydrogen bonds to the amino group in the aspartate structure and the relatively high temperature factors for these atoms in both structures.
...
PMID:Crystal structures of true enzymatic reaction intermediates: aspartate and glutamate ketimines in aspartate aminotransferase. 790 48

The influence of enhancing the supply of hydrogen donors on respiratory rates, NAD(P)H fluorescence, and membrane potential was investigated. Addition of 5 mM malate to mitochondria during oxidation of 10 mM isocitrate, oxoglutarate, succinate, proline, or glycerol-3-phosphate under steady-state conditions resulted in an inhibition of respiration, coincident with a decrease in both transmembrane electrical potential and percentage reduction of NAD(P). Half-maximum inhibition of NAD(P) reduction in the resting state of 10 mM isocitrate respiration was reached at 10 mM malate. This inhibition was concluded to be due to oxaloacetate formed immediately from malate by succinate dehydrogenase. Addition of 5 mM isocitrate caused higher respiratory rates, accompanied by an increase in both delta psi and percentage of NAD(P) reduction, in mitochondria oxidizing 10 mM oxoglutarate, glutamate, proline, hydroxybutyrate, glycerol-3-phosphate, or 0.025 mM palmitoyl carnitine. The half-maximum increase in percentage NAD(P) reduction with 10 mM 2-oxoglutarate as primary substrate was found at 0.24 mM isocitrate. Within the citric acid cycle, succinate dehydrogenase and NAD-isocitrate dehydrogenase play an important role in changes in the rate of NADH formation. Therefore, they participate in flux control. Furthermore, mitochondrial aspartate aminotransferase and oxidoreductases of the beta-oxidation pathway of fatty acids are additionally involved in adjusting the rate of NADH formation.
...
PMID:Contribution to control of mitochondrial oxidative phosphorylation by supplement of reducing equivalents. 791 69

Tyr70 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by oligonucleotide-directed mutagenesis. Aspartate aminotransferase Y70H retained at pH 7.5 13% of the activity toward dicarboxylic amino acids, whereas the activity toward aromatic amino acids was only 0.6% of that of the wild-type enzyme, corresponding to a 22-fold increase in the ratio of the activities toward these two types of substrates. In comparison to that of the wild-type enzyme, the low-pH limb of the pH-activity profile of the mutant enzyme was shifted to higher pH values, very likely reflecting the titration curve of the newly introduced histidine residue with a pKa' of 6.3. Apparently, a positively charged residue at position 70 abolishes enzymic activity. The spectrophotometrically determined pKa' value of the internal aldimine formed between pyridoxal 5'-phosphate and Lys258 in the mutant enzyme was 6.0, similar to that in the wild-type enzyme. The rate constant of the dissociation of pyridoxamine 5'-phosphate from the mutant enzyme was increased only 3 times over that of the wild-type enzyme, in contrast to the 80-fold increase in Escherichia coli aspartate aminotransferase Y70F [Toney, M. D., & Kirsch, J. F. (1987) J. Biol. Chem. 262, 12403-12405], suggesting that His70 can replace Tyr70 in forming a hydrogen bond to the coenzyme.
...
PMID:Shift in pH-rate profile and enhanced discrimination between dicarboxylic and aromatic substrates in mitochondrial aspartate aminotransferase Y70H. 813 Jan 87

The strictly conserved active site residue, Asp222, which forms a hydrogen-bonded salt bridge with the pyridine nitrogen atom of the pyridoxal 5' phosphate (PLP) co-factor of aspartate aminotransferase (AATase), was replaced with alanine (D222A) in the Escherichia coli enzyme. The D222A mutant exhibits non-hyberbolic saturation behavior with amino acid substrates which appear as apparent negative cooperativity in steady-state kinetic analyses. Single turnover progress curves for D222A are well described by the sum of two exponentials, contrasting with the monophasic kinetics of the wild-type enzyme. An active/inactive heterodimer containing the D222A mutation retains this biphasic kinetic response, proving that the observed cooperativity is not the result of induced allostery. The anomalous behavior is explained by a hysteretic kinetic model involving two slowly interconverting enzyme forms, only one of which is catalytically competent. The slow functional transition between the two forms has a half-life of approximately 10 mins. Preincubation of the mutant with the dicarboxylic inhibitor maleate shifts the equilibrium population of the enzyme towards the catalytically active form, suggesting that the slow transition is related to the domain closure known to occur upon association of this inhibitor with the wild-type enzyme. The importance of Asp222 in the chemical steps of transamination is confirmed by the approximately 10(5)-fold decrease in catalytic competence in the D222A mutant, and by the large primary C alpha-deuterium kinetic isotope effect (6.7 versus 2.2 for the wild-type). The transamination activity of the D222A mutant is enhanced 4- to 20-fold by reconstitution with the co-factor analog N-methylpyridoxal-5'-phosphate (N-MePLP), and the C alpha-proton abstraction step is less rate determining, as evidenced by the decrease in the primary kinetic isotope effect from 6.7 to 2.3. These results suggest that the conserved interaction between the protonated pyridine nitrogen of PLP and the negatively charged carboxylate of Asp222 is important not only for efficient C alpha-proton abstraction, but also for conformational transitions concomitant with the transamination process.
...
PMID:Characterization of the apparent negative co-operativity induced in Escherichia coli aspartate aminotransferase by the replacement of Asp222 with alanine. Evidence for an extremely slow conformational change. 817 90

Three crystal structures of wild type E. coli aspartate aminotransferase (E.C.2.6.1.1) in space group P2(1) have been determined at resolution limits between 2.6 and 2.35 A. The unliganded enzyme and its complexes with the substrate analogues maleate and 2-methylaspartate resulted in different conformations. The unit cell parameters of the unliganded and the inhibited enzyme are a = 87.2, b = 79.9, c = 89.8 A and beta = 119.1 degrees, and a = 85.4, b = 79.8, c = 89.5 A and beta = 118.6 degrees, respectively. The crystallographic symmetry is pseudo-C222(1). The liganded enzyme structures were solved by difference Fourier techniques from that of a Val39-->Leu mutant partially refined to an R-factor of 0.22 at 2.85 A. They have a "closed" conformation like the chicken mAATase:maleate complex. The models were refined to R-factors of 0.19 (maleate complex) and 0.18 (2-methylaspartate complex) by molecular dynamics and restrained least squares methods. The unliganded crystal form was solved by molecular replacement and refined to an R-factor of 0.19 at 2.5 A resolution. The structure is in a "half-open" conformation, with the small domain rotated about 6 degrees from the closed conformation. The cofactor pyridoxal phosphate has a more relaxed conformation than in mAATase. Both maleate and 2-methylaspartate are hydrogen-bonded to the active site as in mAATase. The C alpha-CH3 bond of 2-methylaspartate is oriented at right angles to the cofactor pyridine ring, the most productive orientation for alpha-deprotonation of the substrate L-aspartate. Comparisons with earlier determined eAATase structures in space group C222(1) revealed differences that can probably be attributed to the somewhat lower resolution of the orthorhombic structures and/or mutations in the eAATases used in those studies. The present P2(1) structures confirm the justification of extrapolating properties of active site point mutants to the vertebrate isozymes. They will serve as reference in the interpretation of the properties of further site-directed mutants in continued studies of structure-function relationships of this enzyme.
...
PMID:Crystal structures of Escherichia coli aspartate aminotransferase in two conformations. Comparison of an unliganded open and two liganded closed forms. 819 59

Aromatic amino acid aminotransferase (ArAT) from Escherichia coli was overexpressed in E. coli cells, purified, and characterized. The enzyme was similar to aspartate aminotransferase (AspAT) of E. coli in many aspects, such as gross protein structure and spectroscopic properties. The reactions of pyridoxal 5'-phosphate-form ArAT with amino acids and pyridoxamine 5'-phosphate-form ArAT with oxo acids were investigated using stopped-flow spectrophotometric techniques. The kinetic parameters for these "half" reactions could excellently explain the ArAT-catalyzed overall transamination reactions at pH 8.0. Reactions of ArAT with aspartate and tryptophan which had been deuterated at position 2 showed isotope effects of 2.5 and 6.0 in the kcat values of the half-reactions, showing that the proton-transfer step is at least partially rate-limiting for these reactions. ArAT and AspAT showed overlapping substrate specificity. Both ArAT and AspAT were active toward dicarboxylic substrates. ArAT showed, however, 10(3)-fold higher activity toward aromatic substrates than AspAT. This high activity toward aromatic substrates was in part ascribed to the active site hydrophobicity of ArAT, which was suggested to be about 1.4 times as large as that of AspAT. In addition to dicarboxylic substrate analogs, aromatic substrate analogs such as carboxylic acids, 2-methyl amino acids, and 3-hydroxy amino acids caused characteristic changes in the absorption spectra of ArAT, while these aromatic analogs did not significantly change the spectra of AspAT. In particular, the erythro-3-hydroxy analogs of phenylalanine and aspartate caused a prominent absorption of ArAT at around 500 nm, which is generally ascribed to the accumulation of quinonoid intermediates. The threo forms of these 3-hydroxy analogs acted as substrates for ArAT. The erythro and threo forms of 3-hydroxyaspartate reacted with AspAT similarly as they reacted with ArAT; however, both forms of 3-phenylserine were poor substrates for AspAT, although phenylalanine was a fairly good substrate for AspAT. The observations on the two erythro-3-hydroxy amino acids show the similar orientation of these analogs in the active site of ArAT, probably through a hydrogen-bonding network involving the hydroxy groups of the analogs and Tyr70, and suggest that the aromatic binding pocket is near or even overlaps the side-chain-carboxylate-binding site for dicarboxylic substrates.
...
PMID:Escherichia coli aromatic amino acid aminotransferase: characterization and comparison with aspartate aminotransferase. 821

The azomethine (Schiff base) linkage between the epsilon-amino group of active-site lysine 258 and the carbonyl moiety of enzyme-bound pyridoxal 5'-phosphate (PLP) normally exhibits absorbance maxima at ca. 360 (high-pH form) or ca. 430 nm (low-pH form). However, the absorbance maximum is shifted from 358 to 386 nm, a value which is similar to that of free PLP (lambda max = 388 nm), in a mutant form of Escherichia coli aspartate aminotransferase (AATase) in which tyrosine 225, which normally donates a hydrogen bond to the phenolate function of PLP, has been replaced with phenylalanine (Y225F). This spectral shift suggested that PLP binds to Y225F as the free aldehyde. The following evidence from isotope-edited classical Raman spectroscopy proves conclusively that the near-UV spectrum is anomalous and that PLP is bound to Y225F as a Schiff base: (1) A strong cofactor peak at 1630 cm-1 in the holoenzyme-minus-apoenzyme difference spectrum of the unprotonated form of Y225F is red-shifted by 18 cm-1 in enzyme labeled with 15N at lysine 258 and other positions. (2) This isotope-induced red shift is similar to that observed in the unprotonated form of the model Schiff base, PLP-valine. (3) The Raman spectrum of Y225F is unchanged in H(2)18O, while peaks at ca. 1670 cm-1 in the spectrum of free PLP or in that of a mutant of AATase in which Lys-258 is replaced with Ala, are red-shifted by ca. 30 cm-1 in H(2)18O.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Structure of the complex between pyridoxal 5'-phosphate and the tyrosine 225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase determined by isotope-edited classical Raman difference spectroscopy. 834 9

In this article the spontaneous chemiluminescence and the steady-state concentration of hydrogen peroxide were determined in rat liver as indicators of oxidative stress in the tissue. Hydroperoxide-initiated chemiluminescence and the activity of antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) were also measured to evaluate antioxidant defenses and serum activity of lactate dehydrogenase and aspartate aminotransferase. Mitochondrial morphology and mitochondrial respiratory control ratio were measured as indicators of cell and mitochondrial damage. Xanthine dehydrogenase and xanthine oxidase activities were determined as a possible source of oxyradicals. No significant changes were observed after 10 or 30 min of vena cava occlusion in any of the measured parameters. In contrast, 10 min of occlusion followed by 10 min of reperfusion increased chemiluminescence (from 18 +/- 3 to 32 +/- 5 cps/cm2), hydrogen peroxide (from 0.10 +/- 0.01 to 0.17 +/- 0.01 mumol/L), lactate dehydrogenase (from 80 +/- 2 to 330 +/- 30 U/L), and aspartate aminotransferase (from 42 +/- 2 to 100 +/- 10 U/L). Liver reperfusion was also associated with mitochondrial swelling and decreased mitochondrial respiratory control (from 5.6 +/- 0.3 to 2.6 +/- 0.1). The activity of the antioxidant enzymes and xanthine oxidase was instead without change. After 30 min of vena cava occlusion and 10 min of reperfusion a more marked increase in chemiluminescence (37 +/- 5 cps/cm2), hydrogen peroxide (0.30 +/- 0.01 mumol/L), lactate dehydrogenase (730 +/- 10 U/L) and aspartate aminotransferase (140 +/- 10 U/L) was observed. No further changes were found in either mitochondrial morphology or respiratory control (2.4 +/- 0.1) in isolated mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Oxidative stress produced by suprahepatic occlusion and reperfusion. 840 64

<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>