Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P17174 (aspartate aminotransferase)
 14,872 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The pyridoxal form of both cytosolic and mitochondrial aspartate aminotransferase is irreversibly inactivated consequent to its interaction with the beta,gamma-unsaturated substrate analogue vinylglycine. Per catalytic cycle, 90% of the enzyme molecules are inactivated while 10% escape inactivation by transamination to the pyridoxamine form. In the presence of vinylglycine plus 2-oxoglutarate, inactivation is complete because of retransamination of the pyridoxamine form to the susceptible pyridoxal form. Peptide analyses after inactivation with [1-14C]vinylglycine showed that vinylglycine alkylates the active-site lysine residue 258 which forms the internal aldimine with the coenzyme pyridoxal 5'-phosphate. The coenzyme itself is left intact; resolution of the inactivated enzyme by base or trichloroacetic acid yields pyridoxal-5'-P. The absorption spectrum of the inactivated enzyme (lambdamax 335 nm) suggests that the cofactor is bound as a substituted aldimine. The proposed pathway of alkylation of Lys-258 involves abstraction of the alpha proton from vinylglycine, isomerization to the alpha,beta-unsaturated enamine, and subsequent nucleophilic attack of the epsilon-amino group of the lysyl residue at the beta carbon of the inhibitor. The determination of the amino acid sequence around the coenzyme-binding lysyl residue in the mitochondrial isoenzyme from chicken gave Ala-(epsilon-Pxy)Lys-Asn-Met-(Gly,Leu,Tyr) which is identical with the other mitochondrial transaminases examined so far.
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PMID:Active-site labeling of aspartate aminotransferases by the beta,gamma-unsaturated amino acid vinylglycine. 91 93

Holo and apoenzyme of aspartate aminotransferase from beef kidney are 80% inactivated by photoxidation in the presence of 2 X 10(-6) M tetraiodofluroescein with the modification of two histidine residues per enzyme protomer. At a higher concentration (1 X 10(-5) M) a tyrosine residue is also modified. The keto substrates, ketoglutarate and oxalacetate, protect the enzyme from photoxidation. Diethylpyrocarbonate modifies three histidine residues per enzyme protomer and reduces the activity only 10%. These results suggest that the two histidine residues photoxidized through the sensitizer, are located in the active site of the enzyme, at least one of these appears to be involved in ketosubstrate binding. The other three histidines modified by diethylpyrocarbonate are likely located on the enzyme surface and are not involved in the catalytic activity of the enzyme.
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PMID:Chemical modifications of histidine residues in cytoplasmic asparate aminotransferase from beef kidney. 94 May 48

Mitochondrial aspartate aminotransferase from beef kidney is 50% inhibited after 2 hr treatment with 2.5 mM tetranitromethane at pH 8. Two tyrosine residues per enzyme protomer (46,000 daltons) are modified by the reagent either in the holoenzyme or in the apoenzyme. In both cases the five SH groups titratable with p-mercuribenzoate are not modified by the reagent. However, with a tetranitromethane concentration higher than 2.5 mM and 10 mM mercaptoethanol, an additional tyrosine residue is nitrated in both holo- and apoenzymes. These results are not affected by the presence in the incubation mixture of the substrates alpha-ketoglutarate and glutamate both at ten times their Km values. Mercaptoethanol does not impair the recombination of native or nitrated apoenzyme with the coenzyme and does not reduce the coenzyme moiety of native or nitrated holoenzyme, but promotes a conformational change in the nitrated holoenzyme which causes inactivation. Hydrosulfite promotes the reduction of the coenzyme moiety of native and nitro holoenzyme resulting in their inactivation, largely in the nitrated form. The recombination of the coenzyme with native or nitrated apoenzyme is not influenced by hydrosulfite.
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PMID:Role of tyrosine residues in mitochondrial aspartate aminotransferase from beef kidney. 117 45

Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that the active sites of glutamate aspartate transaminase function independently and a compulsory flip-flop mechanism is not involved.
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PMID:Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction. 117 14

The sequences of the coenzyme-binding peptide of both cytoplasmic and mitochondrial aspartate aminotransferases from sheep liver were determined. The holoenzymes were treated with NaBH4 and digested with chymotrypsin; peptides containing bound pyridoxal phosphate were then isolated. One phosphopyridoxyl peptide was obtained from sheep liver cytoplasmic aspartate aminotransferase. Its sequence was Ser-Ne-(phosphopyridoxyl)-Lys-Asn-Phe. This sequence is identical with that reported for the homologous peptide from pig heart cytoplasmic aspartate aminotransferase. Two phosphopyridoxyl peptides with different RF values were isolated from the sheep liver mitochondrial isoenzyme. They had the same N-terminal amino acid and similar amino acid composition. The mitochondrial phosphopyridoxyl peptide of highest yield and purity had the sequence Ala-Ne-(phosphopyridoxyl)-Lys-Asx-Met-Gly-Leu-Tyr. The sequence of the first four amino acids is identical with that already reported for the phosphopyridoxyl tetrapeptide from the pig heart mitochondrial isoenzyme. The heptapeptide found for the sheep liver mitochondrial isoenzyme closely resembles the corresponding sequence taken from the primary structure of the pig heart cytoplasmic aspartate aminotransferase.
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PMID:The sequences of the coenzyme-binding peptide in the cytoplasmic and the mitochondrial aspartate aminotransferases from sheep liver. 118 Aug 94

Cytoplasmic aspartate aminotransferase from beef kidney loses 25% of its activity on nitration with tetranitromethane while the apoenzyme about 95%. In the holoenzyme 0.5 tyrosine residue and 1.0 tyrosine residue in the apoenzyme are nitrated per enzyme protomer. In addition 1 cysteine residue per protomer is oxidized in both. The presence of substrates, alpha-ketoglutarate and glutamate, both at ten times their Km values, does not change these results. Mercaptoethanol does not affect the residual activity of either the nitrated holo or apoenzyme. Dithionite abolishes the activity of the nitrated holoenzyme by reducing tha coenzyme moiety. It has no effect on the native holoenzyme or on either the native or nitroapoenzyme.
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PMID:Role of tyrosine residues in cytoplasmic aspartate aminotransferase from beef kidney. 127 58

The distribution of amino acids between plasma, liver and brain was studied in adult male rats, fed a diet containing 8.7, 17 (control animals), 32 and 51% of protein during 15 days. The caloric intake was nearly equal in all groups. The highest food intake was observed in the animals on the low protein diet. Changes in plasma amino acids were variable. In contrast to the behavior of most amino acids in plasma, the branched chain amino acids were highest in the animals fed the 51% protein diet. Despite the low protein intake in the animals fed a 8.7% protein diet, the concentration of serine, glutamic acid, glutamine, glycine, alanine, methionine, isoleucine, leucine, phenylalanine and ornithine were significantly higher compared to control animals, whereas in those receiving a high protein diet, valine, leucine, tyrosine, tryptophan and histidine increased in relation to the increased protein and amino acid intake. The plasma amino acid patterns are not greatly influenced by the amino acid distribution in the food and the amount ingested. Alanine aminotransferase, aspartate aminotransferase, glutamate dehydrogenase and cholinesterase showed a two- to fivefold increased activity in the liver of animals consuming a high protein diet. In the brain, the concentration of valine, leucine, isoleucine, phenylalanine and tyrosine in animals receiving the low protein diet was higher than in controls and increased further with increasing protein content of the diet. Glutamine was increased in all dietary groups. The predicted influx of amino acids showed increasing influx rates in dependence of the plasma amino acid concentration. The entry of tyrosine and tryptophan and their brain concentration was inversely proportional to the protein content of the diet. In the present study which considers long-term adaptation to an increasing protein and amino acid intake in comparison to a balanced control protein diet, the levels of the indispensable amino acids were maintained within narrow limits in the brain and liver. The results indicate that inspite of a variable protein intake, the body tends to keep organ amino acids in relatively narrow limits favoring in this way amino acid homeostasis.
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PMID:Effect of different protein diets on the distribution of amino acids in plasma, liver and brain in the rat. 159 Jun 69

Aspartate aminotransferase (mitochondrial isoenzyme from chicken) has been found to racemize very slowly dicarboxylic amino acid substrates in the presence of their cognate oxo acids [Kochhar, S. & Christen, P. (1988) Eur. J. Biochem. 175, 433-438]. Tyrosine, phenylalanine and alanine are racemized at the same rate although they undergo the transamination reaction 3-5 orders of magnitude more slowly than the dicarboxylic substrates. Similarly, the truncated enzyme aspartate aminotransferase-(27/32-410) catalyzes the racemization at the same rate as the native enzyme, while its rate of transamination is decreased to 3% of that of the native enzyme. Apparently, the rate-limiting step in racemization is not immediately linked to the transamination cycle. Decreasing the water concentration in the reaction medium by adding methanol at 0 degrees C drastically reduces the rate of racemization without affecting the rate of transamination. On the basis of these and additional kinetic data and the model of the three-dimensional structure of the active site, we conclude that a water molecule is responsible for the protonation of C alpha of the coenzyme-substrate intermediate from the wrong side. The diffusion of the water molecule into the interior of the enzyme appears to be the rate-limiting step in aspartate-aminotransferase-catalyzed racemization.
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PMID:Mechanism of racemization of amino acids by aspartate aminotransferase. 173 41

Absorption and circular dichroism spectra of stable enzyme-substrate intermediates of aspartate aminotransferase were recorded at subzero temperatures (down to -65 degrees C) in the cryosolvent water/methanol. The intermediates were formed either between the pyridoxal form of the enzyme and its amino acid substrates, or between the pyridoxamine form and its oxo acid substrates. Kd values determined by spectroscopic titration were very close to the Km values reported for the different substrates. The adsorption complex of the pyridoxal form was probably obtained on addition of cysteine sulfinate. This complex is characterized by an increased absorption at 430 nm together with a positive Cotton effect, as also observed in the case of the complex with the competitive inhibitor maleate indicating protonation of the internal aldimine. Addition of the substrates aspartate or glutamate to the pyridoxal form seemed to result in the direct accumulation of the external aldimine which showed a slight decrease in both the absorbance and the Cotton effect at 360 nm. Additionally, a bathochromic shift of 5 nm was observed in the case of glutamate. At 430 nm, only a minor increase in absorbance, but not in circular dichroism, was observed with aspartate, and no changes were found with glutamate and the substrate analog 2-methylaspartate, indicating a deprotonated external aldimine. Presumably, the ketimine intermediate was obtained on addition of the oxo acids 2-oxoglutarate or oxalacetate to the pyridoxamine form. The intermediate showed a slight bathochromic shift (2 nm) of the absorption band and decreased circular dichroism. On formation of the ketimine, a tyrosine residue, probably active-site Tyr225, becomes partly ionized. The finding that the external aldimine can probably be accumulated in the conversion of the pyridoxal to the pyridoxamine form with the natural substrates would confirm the proton abstraction at C alpha to be the rate-limiting step in the tautomerization, although with cysteine sulfinate, the formation of the external aldimine might contribute to the rate limitation. Accumulation of the ketimine in the reverse direction would indicate that the proton abstraction at C4' is rate-limiting in this half-reaction. The results demonstrate the feasibility of further structural investigations of true enzyme-substrate intermediates.
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PMID:Spectroscopic characterization of true enzyme-substrate intermediates of aspartate aminotransferase trapped at subzero temperatures. 193 64

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus binds pyridoxal 5' phosphate, via an aldimine bond, with Lys-241. This residue has been identified by reducing the enzyme in the pyridoxal form with sodium cyanoboro[3H]hydride and sequencing the specifically labeled peptic peptides. The amino acid sequence centered around the coenzyme binding site is highly conserved between thermophilic aspartate aminotransferases and differs from that found in mesophilic isoenzymes. An alignment of aspartate aminotransferase from Sulfolobus solfataricus with mesophilic isoenzymes, attempted in spite of the low degree of similarity, was confirmed by the correspondence between pyridoxal 5' phosphate binding residues. Using this alignment it was possible to insert the archaebacterial aspartate aminotransferase into a subclass, subclass I, of pyridoxal 5' phosphate binding enzymes comprising mesophilic aspartate aminotransferases, tyrosine aminotransferases and histidinol phosphate aminotransferases. These enzymes share 12 invariant amino acids most of which interact with the coenzyme or with the substrates. Some enzymes of subclass I and in particular aspartate aminotransferase from Sulfolobus solfataricus, lack a positively charged residue, corresponding to Arg-292, which in pig cytosolic aspartate aminotransferase interacts with the distal carboxylate of the substrates (and determines the specificity towards dicarboxylic acids). It was confirmed that aspartate aminotransferase from Sulfolobus solfataricus does not possess any arginine residue exposed to chemical modifications responsible for the binding of omega-carboxylate of the substrates. Furthermore, it has been found that aspartate aminotransferase from Sulfolobus solfataricus is fairly active when alanine is used as substrate and that this activity is not affected by the presence of formate. The KM value of the thermophilic aspartate aminotransferase towards alanine is at least one order of magnitude lower than that of the mesophilic analogue enzymes.
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PMID:The active site of Sulfolobus solfataricus aspartate aminotransferase. 195 27


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