Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.6.1.1 (aspartate aminotransferase)
 21,665 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Spin-labeled analogues of vitamin B6: 2, 2, 6, 6-tetramethyl-N-oxylpiperydinyl-4-(5' phosphopyridoxyl)-amine (1) and 2, 2, 6, 6-tetramethyl-N-oxyl-piperydinyl-4-(pyridoxal-5')-phosphate (II) are synthesized. There analogues were shown to interact in the equimolar ratio with the active site of cytosol aspartate transaminase. It was proved by CD-titration of apotransaminase with I and II and by competition between the coenzyme and synthesized analogues. The free valency of spin-labeled coenzymes immediately disappears after interaction with the apoenzyme due to iminoxyl group reduction. The binding of I and II with the apoenzyme is accompanied by oxidation of one of the inner cysteine residues. The reactivation of the modified apoenzyme with PLP is not less than 65% of original transaminase activity. The analysis of space-filling atomic models of synthesized compounds allows to conclude that the distance between the centre of pyridine ring of the coenzyme and the modified thiol group is not more than 8 A.
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PMID:[Interaction of spin-labeled analogues of vitamin B 6 with the active site of apotransaminase]. 17 69

Using purified enzymes of human origin and patients' sera, we examined factors influencing the in vitro association of pyridoxal phosphate with aspartate aminotransferase (EC 2.6.1.1). The rate of association was markedly retarded by phosphate buffer in comparison with tris(hydroxymethyl)aminomethane or six other buffers. Pyridoxal phosphate at an incubation concentration of 130 mumol/liter reactivated the entire apoenzyme portion of an apoenzyme/holoenzyme mixture within 5 min in tris(hydroxymethyl)aminomethane; in contrast, less than 20% was associated during 15 min in phosphate. Activity measured in tris(hydroxymethyl)aminomethane-buffer without exogenous pyridoxal phosphate was 4% greater than that in phosphate and was slightly increased by increasing the pH of the assay mixture from 7.5 to 8.0. Aspartate in the incubation medium did not retard the stimulation in tris(hydroxymethyl)aminomethane buffer. While the magnitude of stimulation varied greatly among sera, a consistent mean stimulation of 30% for groups of sera with normal activities was found when asparate at 125 mmol/liter, 2-oxoglutarate at 6.7 mmol/liter and tris(hydroxymethyl)aminomethane at 90 mmol/liter were used, an increase over the 16% with phosphate buffer [Clin. Chem. 19, 92 (1973)]. Absorbance spectra suggest pyridoxal phosphate exists as the Schiff base of tris(hydroxymethyl)aminomethane or aspartate, or both, under conditions of assay incubation (without addition of 2-oxoglutarate). Nonenzymatic catalysis of the reaction by pyridoxal phosphate alone or a formation of a protein/pyridoxal phosphate adduct was discounted with use of a D-asparate substrates.
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PMID:Effects of buffers on aspartate aminotransferase activity and association of the enzyme with pyridoxal phosphate. 24 May 13

A simple and convenient procedure is described for the isolation in good yield of two amino-transferases from various strains of Escherichia coli. On the basis of their substrate specificities one of the enzymes has been classified as an aromatic amino acid aminotransferase and the other as an aspartate aminotransferase, but both act on a wide range of substrates. Pyridoxal phosphate is bound more strongly to the aspartate aminotransferase than to the aromatic amino transferase which cannot be fully re-activated after removal of the prosthetic group. Both enzymes are composed of two subunits which appear to be identical.
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PMID:The purification and properties of the aspartate aminotransferase and aromatic-amino-acid aminotransferase from Escherichia coli. 35 93

Aspartate aminotransferase (EC 2.6.1.1) activities in sera from nine healthy individuals were monitored during two weeks, both with and without first supplementing the serum with pyridoxal phosphate. Pyridoxal phosphate supplementation caused a mean increase of 39% (range, 33-55%) in measured activity. The biological variability during the two-week period was independent of pyridoxal phosphate supplemantation. The intra-individual variability (CV) was 5.3% and 5.1% with and without pyridoxal phosphate supplementation, respectively; the corresponding inter-individual variability was 13.2% and 13.6%. We conclude that the reference interval will be insensitive to intra-individual fluctuations in aspartate aminotransferase activity in serum, whether or not the serum is supplemented with pyridoxal phosphate.
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PMID:Biological variability in aspartate aminotransferase activity in serum of healthy persons, and effect of in vitro supplemantation with pyridoxal 5-phosphate. 83 43

Interaction of aspartate aminotransferase with various aminooxycelluloses capable of reacting with carbonyl compounds to form oximes has been studied with respect to the distance of the H2NO-groups from the polymer matrix. Aminotransferase does not react with aminooxycelluloses, when the amono-oxygroups are located at small distances from the matrix. When these celluloses interact with the enzyme in the presence of the substrate amino acid, we manage to obtain the amino-form of amino-transferase containing no pyridoxylidine form quantitatively. The cellulose with the H2NO-groups located at considerable distances from the polysaccharide matrix, form the "aminotransferase oxime--aminooxycellulose" complex, which is subsequently split either to a choloenzyme or apoenzyme of PLP and aminooxycellulose, depending on the experimental conditions used. The adsorbents under study may present some interest both in terms of isolation and purfication of pyridoxal enzymes and their effects on enzymatic systems, which contain carbonylic compounds.
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PMID:[Interaction of aspartate aminotransferase with aminooxyalkylcelluloses]. 85 23

The Y70F mutant of aspartate aminotransferase has reduced affinity for coenzymes compared to the wild type. The equilibrium dissociation constants for pyridoxamine phosphate (PMP) holoenzymes, KPMPdiss, were determined from the association and dissociation rate constants to be 1.3 nM and 30 nM for the wild type and mutant, respectively. This increase in KPMPdiss for Y70F is due to a 27-fold increase in the dissociation rate constant. Pyridoxal phosphate (PLP) association kinetics are complex, with three kinetic processes detectable for wild type and two for Y70F. A directly determined, accurate value of KPLPdiss for wild type enzyme has been difficult to obtain because of the low value of this constant. The values of KPLPdiss for the holoenzymes were determined indirectly through the measured values for KPMPdiss, glutamate-alpha-ketoglutarate half-reaction equilibrium constants, and the equilibrium constant for the transamination of PLP by glutamate catalyzed by Y70F. The values of KPLPdiss obtained by this procedure are 0.4 pM for wild type and 40 pM for Y70F. The increases in KPMPdiss and KPLPdiss for Y70F correspond to delta delta G values of 1.9 and 2.7 kcal/mol, respectively, and are directly attributed to the loss of the hydrogen bond from the phenolic hydroxyl group of Tyr70 to the coenzyme phosphate. The delta G for association of PLP with wild type enzyme is 4.7 kcal/mol more favorable than that for PMP.
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PMID:Kinetics and equilibria for the reactions of coenzymes with wild type and the Y70F mutant of Escherichia coli aspartate aminotransferase. 167 70

Pyridoxal 5'-phosphate is a coenzyme for a number of enzymes which catalyse reactions at C alpha of amino acid substrates including transaminases, decarboxylases and serine hydroxymethyltransferase. Using the X-ray coordinates for a transaminase, aspartate aminotransferase, and the results of stereochemical and mechanistic studies for decarboxylases and serine hydroxymethyltransferase, an active-site structure for the decarboxylase group is constructed. The structure of the active-site is further refined through active-site pyridoxyllysine peptide sequence comparison and a 3-D catalytic mechanism for the L-alpha-amino acid decarboxylases is proposed. The chemistry of serine hydroxymethyltransferase is re-examined in the light of the proposed decarboxylase mechanism.
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PMID:A comparison of pyridoxal 5'-phosphate dependent decarboxylase and transaminase enzymes at a molecular level. 176 22

Pyridoxamine 5'-phosphate in 18 microliters of human capillary blood plasma is determined by catalytic amplification using the apoenzyme of aspartate aminotransferase. Prior isolation from interfering substances is accomplished by employment of a cation exchange resin in batch operation. The procedure consists of the following stages. Stage I, denaturation of proteins. Trichloroacetic acid is used to precipitate plasma proteins and liberate any bound coenzyme. Dilute NaCl is added to expand the volume thus minimizing coenzyme entrapment in the precipitate. Stage II, isolation of the coenzyme. A sulfonated polystyrene ion exchange resin is used inside a centrifugal filter. Pyridoxamine 5'-phosphate in the supernatant from Stage I adsorbs to the resin. Pyridoxal 5'-phosphate, other organic phosphates, and Pi are removed by centrifugation. Rinsing with dilute NaBH4 destroys traces of pyridoxal 5'-phosphate and washes off residual inhibitors. Pyridoxamine 5'-phosphate is then desorbed with NaOH and Tris buffer and recovered by centrifugation. Stage III, reconstitution and assay. The desorbate from Stage II is incubated with excess apoenzyme. Specific activity of the reconstituted enzyme is measured. Interpolation from a standard curve relating enzyme specific activity and pyridoxamine 5'-phosphate concentration yields the plasma level of the cofactor. Approximately 3 h are required to carry out the procedure. Much of the coenzyme was found not be assayable if plasma was refrigerated overnight or if whole blood was left standing at room temperature for a few hours. The degradation was arrested with freezing at -80 degrees C. In a 13-day experiment involving a healthy subject, sharp rises of plasma pyridoxamine 5'-phosphate were found to occur in response to small doses of oral vitamin B6.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Determination of pyridoxamine 5'-phosphate in human blood plasma. 180 57

The coenzyme (PLP) binding domain (residues 47-329) of the dimeric aspartate aminotransferase from Escherichia coli was produced separately by recombinant DNA methods. It folded autonomously both in vivo and in vitro, that is, independently of the native N- and C-terminal extensions that combine to form the small domain of eAAT. The PLP-domain had one binding site for PLP of relatively high affinity involving a covalent bond to the protein. It was monomeric, although the major subunit-subunit interface at the 2-fold symmetry axis remained unchanged. This effect appears to be due mainly to the absence of the N-terminal extension that contains hydrophobic residues, which interact with the PLP-domain of the second subunit in the wild-type dimer. Judged by circular dichroism, fluorescence, and HPLC gel filtration at increasing concentrations of guanidinium chloride, the PLP-domain underwent a three-state unfolding transition (M' in equilibrium M'* in equilibrium U') involving a compact intermediate M'*. This behavior parallels the unfolding of the dissociated native monomer of cAAT.
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PMID:Autonomous folding and coenzyme binding of the excised pyridoxal 5'-phosphate binding domain of aspartate aminotransferase from Escherichia coli. 201 18

The formation of pyridoxal and its phosphate from pyridoxamine phosphate by red cell haemolysates was measured in a centrifugal analyser by the formation of the fluorescent adduct with semicarbazide. Pyridoxal phosphate was found to react more rapidly than pyridoxal, thus permitting a distinction between the two products, and hence the measurement of phosphatase activity. Activity of the enzyme, pyridoxamine phosphate:oxygen oxidoreductase (deaminating) EC 1.4.3.5 (PPO) was measured in haemolysates from 72 Gambian women with evidence of riboflavin deficiency, and was repeated after 6 weeks of placebo or riboflavin supplementation. Those who received the riboflavin supplement responded with a marked increase in PPO activity, which was matched by a decrease in the activation coefficient (AC) of erythrocyte NAD(P)H2:glutathione oxidoreductase, EC 1.6.4.2 (glutathione reductase, EGR). No difference between the supplemented and unsupplemented groups was observed in the capacity of haemolysates to hydrolyse pyridoxal 5-phosphate, nor in the extent of activation of erythrocyte L-aspartate:2-oxoglutarate aminotransferase EC 2.6.1.1. by pyridoxal phosphate. Although the three subjects with low levels of D-glucose 6-phosphate: NADP 1-oxidoreductase EC 1.1.1.49 (G6P-D) had, as expected, correspondingly low AC's of EGR, their unsupplemented activities of PPO were in the same low range as those of the G6P-D-normal subjects, and they responded as G6P-D-normal subjects did to riboflavin supplementation. PPO thus does not appear to resemble EGR in retaining its flavin coenzyme during riboflavin depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A simple fluorimetric assay for pyridoxamine phosphate oxidase in erythrocyte haemolysates: effects of riboflavin supplementation and of glucose 6-phosphate dehydrogenase deficiency. 401 61


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