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

Catalysis-linked conformational transitions of aspartate aminotransferase (cytosolic isoenzyme from pig heart; L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) have been probed by infrared spectrophotometric measurement of hydrogen-deuterium exchange. In the unliganded pyridoxal form of the enzyme at pH 6.0 and 20 degrees, 43% of the total 411 peptide hydrogens per subunit exchange within the first 10 min. An additional 9% exchange slowly in the following time period to 360 min. A quite similar exchange curve is obtained with the pyridoxamine form of the enzyme, indicating close correspondence in conformation of both unliganded forms of the enzyme. Formation of a nonproductive adsorption complex of the pyridoxal enzyme with 2-oxoglutarate or of the pyridoxamine enzyme with glutamate alters the exchange characteristics only slightly. In contrast, the formation of an equilibrium mixture of the covalent transamination intermediates, which occurs in the silultaneous presence of the amino acid and the keto acid substrate, results in a marked retardation of hydrogen exchange, reflecting a substantial tightening of the structure of the enzyme. The exchange reactions of at least 26 peptide hydrogens per subunit (6% of the total) are retarded by a factor of 6 on the average. The occurrence of such syncatalytic conformational changes reflects energetic coupling of the covalency changes at the active site with conformational changes of the macromolecular protein matrix that may contribute to optimizing the free energy profile of enzymic transamination.
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PMID:Syncatalytic conformational changes in aspartate aminotransferase determined by hydrogen-deuterium exchange. 27 28

Escherichia coli aspartate aminotransferase was exposed to aspartate or phenylalanine without oxo acid in buffered 2H2O. The alpha-hydrogen of the amino acids underwent first-order exchange with respect to both substrate and enzyme. P.m.r. spectroscopy gave consistent reaction-rate constants. The deuterium-exchange rate was only moderately increased by addition of oxo acids and was of the same order as the transamination rate. No beta-deuteration was observed. The C(alpha)-H-bond-breaking step is discussed as a part of the entire transamination mechanism.
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PMID:Kinetic studies with the use of proton-magnetic-resonance spectroscopy of the specific alpha-deuteration of amino acids by Escherichia coli aspartate aminotransferase. 35 42

Proton incorporation at position C4 of the substrate-coenzyme Schiff base of aspartate transaminase is a stereospecific process. After carbamylation of the active site Lys-258, the stereospecificity of the reaction in 2H2O is retained. By a correlation method, it is shown that addition occurs from the si side of the complex and the pyridoxamine phosphate produced is deuterated at position pro-S of the pyridoxamine methylene group. These results constitute a demonstration for the stereochemstry of a half-transamination process of the phosphorylated coenzyme under single turnover conditions. They also illustrate that free Lys-258 is not required to maintain stereospecificity and cast doubts on the implication of this residue as a participant in C4 proton addition during catalysis by the native form of this mammalian enzyme.
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PMID:Stereochemistry of holoaspartate transaminase after modification of the active site Lys-258. 42 38

Isolated mitochondria of pigeon and guinea pig liver were subjected to zonal centrifugation. With pigeon liver mitochondria there was uniform distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, aspartate aminotransferase and glutamate dehydrogenase activities. Guinea pig liver mitochondria demonstrated two pyruvate carboxylase and phosphoenolpyruvate carboxykinase maxima but only one maximum with aspartate aminotransferase, malate dehydrogenase and glutamate dehydrogenase. Mitochondrial enzyme levels in rat, pigeon and guinea pig indicate different roles of certain gluconeogenic enzymes in the transport of carbon and hydrogen in and out of mitochondria.
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PMID:The relationship between mitochondrial heterogeneity and gluconeogenesis in liver mitochondria of the rat, pigeon and guinea pig. 119 37

Pulsed Fourier transform proton magnetic resonance spectroscopy was used to study the glutamate-alanine transaminase-catalyzed incorporation of deuterium from solvent deuterium oxide into the alpha and beta positions of L-alanine. It was found that the beta proton resonance signal initially disappears slightly faster than the signal due to the alpha proton, but whereas the alpha proton signal decays exponentially, that due to the beta proton signal does not. Eventually, the rate of decrease of the alpha proton signal becomes greater than that for the beta proton. This change in the relative rates is ascribed to a deuterium isotope effect upon substitution of an alpha proton by a deuteron. Furthermore, as deuterium begins to replace hydrogen, two classes of alanine become distinguishable, i.e. alanine which contains deuterium in the alpha position and hydrogen in the beta position, and alanine which contains hydrogen in the alpha position and deuterium in the beta position. Thus, removal of all 3 beta protons is not contingent upon loss of an alpha proton from the same molecule. The two classes of deuterated alanine may conceivably arise by a scrambling mechanism in which protons are transferred from the alpha to the beta position and vice versa. Present evidence excludes this scramblong mechanism and leads to the conclusion that deuterium incorporation into L-alanine involves, (a) the reversible enzymatic conversion of the classical ketimine enzymes intermediate to an enaminetype structure, and (b) considerable conservation of label during the prototropic shift from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate. It is also postulated that alanine binds at the active site in such a way as to bring the beta protons into close contact with a basic group on the enzyme surface. This group is distinct from that used in abstraction of an alpha proton. The beta protons of glutamate are not enzymatically removed; presumably glutamate binds in such a way that the beta protons cannot effectively interact with an enzyme base. Similar studies were carried out on soluble glutamate-aspartate transaminase; no evidence was found for significant enzyme-catalyzed deuterium incorporation into the beta position of L-glutamate, L-aspartate, and L-alanine.
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PMID:Proton magnetic resonance studies of glutamate-alanine transaminase-catalyzed deuterium exchange. Evidence for proton conservation during prototropic transfer from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate. 124 68

We determined transaminases in human blood serum with an amperometric glutamate biosensor. The probe was a hydrogen peroxide sensor assembled with appropriate selective membranes to enhance the probe specificity and lifetime. Calibration curves of glutamate were linear in the range 1-1000 mumol/L, with a response time of < 1 min. This probe was subsequently applied to the measurement of activities of aspartate and alanine aminotransferases in human sera. Analytical recovery studies demonstrated the suitability of the glutamate sensor by measuring 91-99% of added glutamate, 92-106% of added aspartate aminotransferase, and 101-105% of added alanine aminotransferase. Transaminase activity measured in 80 sera correlated well with results obtained with a spectrophotometric procedure.
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PMID:Analysis for transaminases in serum with an amperometric glutamate electrode. 135 81

The ionization state of the phosphate group bound at the aspartate aminotransferase apoenzyme's active site has been investigated utilizing Fourier-transform infrared spectroscopy following the band corresponding to the symmetric stretching of the dianionic phosphate. Unlike free phosphate, when inorganic phosphate is bound at the enzyme's active site, the integrated intensity value of the dianionic band does not change with pH within the studied range, and this value is similar to that for free dianionic phosphate at pH 8.3. From these results, we propose a dianionic state for the phosphate ion bound to cytosolic aspartate aminotransferase throughout the pH range of 5.7-8.3. The presence of other anions such as acetate and chloride or the substrate aspartate and its analogues produces a pH-dependent phosphate removal from the active site which is favored at low pH values. Elimination of the charged primary amine at the active-site Lys-258, through formation of a Schiff base with pyridoxal or chemical modification by carbamylation, also produces a pH-independent phosphate release. These results are interpreted as Lys-258 together with the active-site alpha-helix and other residues may be involved in stabilizing phosphate as a dianion in the apoenzyme phosphate pocket which anchors the phosphate ester of pyridoxal phosphate in the holoenzyme. It is proposed that the dianionic phosphate contributes to the apoenzyme's thermal stability through formation of strong hydrogen bond and salt bridges with the amino acid residues forming the phosphate binding pocket with assistance of Lys-258, and other active-site cationic components.
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PMID:Inorganic phosphate binding and electrostatic effects in the active center of aspartate aminotransferase apoenzyme. 154 11

The hepatotoxic effects of hyperthermia have been proposed to be related to lipid peroxidation as a consequence of oxidative stress. This can result from exposure of the cell to "radical oxygen" species such as the superoxide and hydrogen peroxide generated by the activity of the oxidase form (type O) of xanthine oxidase (XO), which is converted to that form by perfusion of the liver at hyperthermic temperatures. These radical species are not reactive enough in themselves to cause cell damage but require the presence of a catalyst such as low molecular weight chelated iron. In these studies, ferritin was shown to be a source of iron for the oxidative stress of hyperthermia. (a) Iron was released from ferritin in vitro by the activity of rat liver XO. The rate of iron release from ferritin in this incubation system was a function of the amount of type O XO present and the temperature. Inclusion of allopurinol or superoxide dismutase in the incubation resulted in significantly lower rates of iron release. (b) Livers from Sprague-Dawley rats were perfused at 42.5 degrees and 37 degrees C for 1 h. During the recirculating perfusion, loss of iron from the liver into the perfusate was significantly greater (P less than 0.05) at 42.5 degrees C than at 37 degrees C. Also, there was a pronounced increase in the lactate dehydrogenase and aspartate aminotransferase enzymes in the perfusate during perfusion at 42.5 degrees C. Furthermore, intrahepatic levels of low molecular weight chelated iron were significantly (P less than 0.05) increased following perfusion at 42.5 degrees C. All these responses were abrogated by the inclusion of allopurinol in the perfusate. (c) Oxidative stress, assessed by the efflux of glutathione and oxided glutathione from the liver at 42.5 degrees and 37 degrees C, was significantly (P less than 0.05) increased at the hyperthermic temperature. This oxidative stress was inhibited by iron chelation and allopurinol. These results demonstrate that there is a causal relationship between the generation of superoxide by type O XO produced by hyperthermic perfusion and mobilization of iron from ferritin to form a pool of low molecular weight chelated iron. This iron pool in combination with active oxygen species leads to oxidative stress and lipid peroxidation.
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PMID:Involvement of xanthine oxidase in oxidative stress and iron release during hyperthermic rat liver perfusion. 155 Oct 99

Observation of the 93-kDa cytosolic aspartate aminotransferase by 500-MHz 1H NMR spectroscopy in H2O has revealed a series of resonances in the 10-18 ppm range arising from exchangeable protons. One of these (peak A) has been assigned to the proton bound to the ring nitrogen of the coenzyme pyridoxal 5'-phosphate. A second (peak B) is assigned to H143 which participates in a chain of hydrogen bonds that includes also the coenzyme-bound proton. There is a mutual nuclear Overhauser effect between these two resonances. Peaks A and B respond to changes in pH and to interaction of the enzyme with coenzyme derivatives and inhibitors. Peak A moves from 15.4 to 17.4 ppm as the pH is lowered, while peak B moves in the opposite direction from 14.7 to 13.7 ppm, both with an apparent pKa of 6.15. This pKa is associated with deprotonation of the imine nitrogen at the Schiff base linkage of the coenzyme with K258 of the enzyme. In spectra of enzyme containing pyridoxamine 5'-phosphate, peak A is observed at 16.5 ppm and peak B is at 13.9 ppm over a broad pH range. Peaks A and B are found at 17.8 and 14.0 ppm, respectively, for the enzyme complex with glutarate. When alpha-methylaspartate is added to the enzyme several new resonances appear in the spectrum, which are attributed to formation of the external aldimine. The position of peak A in spectra of various forms of the enzyme is interpreted to reflect the electronic distribution in the coenzyme ring. Several other peaks in this region of the spectrum also are sensitive to changes in pH or the addition of inhibitors. Some possible assignments of these resonances are discussed.
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PMID:NMR observation of exchangeable protons of pyridoxal phosphate and histidine residues in cytosolic aspartate aminotransferase. 165 26

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


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