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At least 19 recordsLinked to original sources
PubMed1991

The active site of Sulfolobus solfataricus aspartate aminotransferase.

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.

Amino Acid Sequence↗
PubMed1995

Evolutionary analysis of aspartate aminotransferases.

Aspartate aminotransferase isoenzymes are located in both the cytosol and organelles of eukaryotes, but all are encoded in the nuclear genome. In the work described here, a phylogenetic analysis was made of aspartate aminotransferases from plants, animals, yeast, and a number of bacteria. This analysis suggested that five distinct branches are present in the aspartate aminotransferase tree. Mitochondrial forms of the enzyme form one distinct group, bacterial aspartate aminotransferase formed another, and the plant and vertebrate cytosolic isoenzymes each formed a distinct group. Plant cytosolic isozymes formed a further group of which the plastid sequences were a member. The yeast mitochondrial and cytosolic aspartate aminotransferases formed groups separate from other members of the family.

Amino Acid Sequence↗
PubMed2003

How does an enzyme evolved in vitro compare to naturally occurring homologs possessing the targeted function? Tyrosine aminotransferase from aspartate aminotransferase.

Aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase) are Escherichia coli paralogs that share 43% sequence identity. A plausible model posits that TATase arose from a duplication of an ancestral AATase-like enzyme. Directed evolution of AATase to an enzyme having TATase activity was undertaken in order to compare the evolved AATase variants with homologous TATases. Eight rounds of DNA shuffling and in vivo selection followed by a backcross with WT AATase produced enzymes that exhibited 100-270-fold increases in k(cat)/K(m)(Phe) and had as much as 11% of the tyrosine aminotransferase activity of WT E.coli TATase. Amino acid substitutions in 11 clones from rounds 7 and 8 were compared with conserved residues in AATases and TATases. The findings are conveniently and compactly illustrated by the use of Venn diagrams and set theory notation. A statistically significant (0.001 or=75% identical) in AATases and variable (<75% identical) in TATases. Very few mutations occur in the intersection (set AAT intersection TAT) of amino acid residues that are conserved in both enzyme types. Seven mutations from set AAT-TAT were combined by site-directed mutagenesis to give a construct that is 60% as active as the best round 8 enzyme, which has 13 amino acid replacements. The Venn diagrams may provide a generally useful tool to highlight the most important specificity determinants for rational redesign. Amino acid replacements were mapped onto the crystal structure of a hydrocinnamate complex of a designed TATase. Five of the seven positions most frequently substituted in the evolved clones are within 15 A of the phenyl side-chain, but only six of the 48 positions that were mutated once or twice are within that radius. Context dependence, neutral mutations, different selective pressures, and stochastic components provide explanations for the observation that many of the substitutions found in the directly evolved enzymes differ from the corresponding amino acids found in the modern natural TATases.

Amino Acid Sequence↗
PubMed1984

Selective inhibition of alanine aminotransferase and aspartate aminotransferase in rat hepatocytes.

Experiments were conducted with intact rat hepatocytes to identify inhibitors and incubation conditions that cause selective inhibition of alanine aminotransferase or aspartate aminotransferase. Satisfactory results were obtained by preincubating cells with L-cycloserine or L-2-amino-4-methoxy-trans-but-3-enoic acid in the absence of added substrates. When cells were incubated for 20 min with 50 microM-L-cycloserine, alanine aminotransferase activity was decreased by 90%, whereas aspartate aminotransferase was inhibited by 10% or less. On subsequent incubation, synthesis of glucose and urea from alanine was strongly inhibited, but glucose synthesis from lactate was unaffected. L-2-Amino-4-methoxy-trans-but-3-enoic acid (400 microM) in hepatocyte incubations caused 90-95% inactivation of aspartate aminotransferase, but only 15-30% loss of alanine aminotransferase activity. After preincubation with the inhibitor, glucose synthesis from lactate was almost completely blocked; with alanine as the substrate, gluconeogenesis was unaffected, and urea synthesis was only slightly decreased. By comparison with preincubation with inhibitors, simultaneous addition of substrates (alanine; lactate plus lysine) and inhibitors (cycloserine; aminomethoxybutenoic acid) resulted in smaller decreases in aminotransferase activities and in metabolic rates. Other compounds were less satisfactory as selective inhibitors. Ethylhydrazinoacetate inactivated the two aminotransferases to similar extents. Vinylglycine was almost equally effective in blocking the two enzymes in vitro, but was a very weak inhibitor when used with intact cells. Concentrations of DL-propargylglycine (4 mM) required to cause at least 90% inhibition of alanine aminotransferase in hepatocytes also caused a 16% decrease in aspartate aminotransferase. When tested in vitro, alanine aminotransferase was, as previously reported by others, more sensitive to inhibition by amino-oxyacetate than was aspartate aminotransferase, but in liver cell incubations the latter enzyme was more rapidly inactivated by amino-oxyacetate.

Alanine Transaminase↗
PubMed1995

Nucleotide sequence of a cDNA coding for bovine mitochondrial aspartate aminotransferase.

Aspartate aminotransferase is a pyridoxal-phosphate dependent enzyme which plays a key role in cell metabolism. We describe the cloning and sequence analysis of the cDNA encoding bovine mitochondrial aspartate aminotransferase and compare the sequence with those of isoenzymes from other mammalian species. An adult bovine heart cDNA library constructed in lambda lambda gt11 was screened using two 32P-end labeled synthetic oligonucleotides. From the screening of the cDNA library two positive clones were isolated. A subclone in pEMBL18, 6B2, generated from the longest recombinant phage was further analyzed. This clone contains an insert of 2500 bp with an Open Reading Frame of 1287 bp that encodes a protein of 430 amino acids. The deduced amino acid sequence confirms previous results obtained by mass spectrometric sequencing. We calculated the percentage of amino acid identity for each protein pair and for each comparison the average number of amino acid substitution per site (Kaa); the lowest Kaa values were obtained from the comparison between the bovine and pig enzymes. This study shows that the rate of evolution of mammalian mitochondrial AspAT is lower and more constant than the equivalent cytosolic enzyme and adds to the growing body of knowledge on the evolution of the aspartate aminotransferase.

Amino Acid Sequence↗
PubMed1992

Mechanism of racemization of amino acids by aspartate aminotransferase.

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.

Amino Acids↗
PubMed1979

Alanine aminotransferase and aspartate aminotransferase measurements with two automated analyzers, SMAC and the ABA-100, compared.

Measurements of alanine aminotransferase and aspartate aminotransferase with the SMAC were evaluated for correlation with the ABA-100, precision, linearity, and carryover. We assayed 200 specimens with normal and abnormal aminotransferase activities with both the SMAC (y) and the ABA-100 (X). Linear regression analysis of the data yielded the following: alanine aminotransferase (r = 0.9732, y = 0.96x + 3.8); and aspartate aminotransferase (r = 0.9892, y = 0.90x + 2.1). Both aminotransferases demonstrated acceptable intra- and inter-assay variations with the SMAC and ABA-100. With the SMAC the upper limit of linearity for alanine aminotransferase was 350 U/L; that for asparate aminotransferase was 450 U/L. Carryover studies for SMAC indicate that specimens immediately following specimens with alanine aminotransferase activities greater 400 U/L and (or) aspartate aminotransferase activities greater than 500 U/L should be re-analyzed.

Alanine Transaminase↗
PubMed1989

Evolutionary relationships among aminotransferases. Tyrosine aminotransferase, histidinol-phosphate aminotransferase, and aspartate aminotransferase are homologous proteins.

A data base was compiled containing the amino acid sequences of 12 aspartate aminotransferases and 11 other aminotransferases. A comparison of these sequences by a standard alignment method confirmed the previously reported homology of all aspartate aminotransferases and Escherichia coli tyrosine aminotransferase. However, no significant similarity between these proteins and any of the other aminotransferases was detected. A more rigorous analysis, focusing on short sequence segments rather than the total polypeptide chain, revealed that rat tyrosine aminotransferase and Saccharomyces cerevisiae and Escherichia coli histidinol-phosphate aminotransferase share several homologous sequence segments with aspartate aminotransferases. For comparison of the complete sequences, a multiple sequence editor was developed to display the whole set of amino acid sequences in parallel on a single work-sheet. The editor allows gaps in individual sequences or a set of sequences to be introduced and thus facilitates their parallel analysis and alignment. Several clusters of invariant residues at corresponding positions in the amino acid sequences became evident, clearly establishing that the cytosolic and the mitochondrial isoenzyme of vertebrate aspartate aminotransferase, E. coli aspartate aminotransferase, rat and E. coli tyrosine aminotransferase, and S. cerevisiae and E. coli histidinol-phosphate aminotransferase are homologous proteins. Only 12 amino acid residues out of a total of about 400 proved to be invariant in all sequences compared; they are either involved in the binding of pyridoxal 5'-phosphate and the substrate, or appear to be essential for the conformation of the enzymes.

Amino Acid Sequence↗
PubMed1976

Influence of auxiliary enzymes on the spectrophotometric measurement of alanine aminotransferase and aspartate aminotransferase activities.

We investigated the enzyme activity of the blank in the spectrophotometric determination of the aminotransferase activities and aspartate aminotransferase activity. 6 lactate dehydrogenase and 3 malate dehydrogenase preparations from different manufactures and from different organs showed additional and contaminating activity. The additional activity depends upon the 2-oxoglutarate concentration. The contaminating activity is caused by alanine aminotransferase and aspartate aminotransferase in the auxiliary enzymes. We propose that exact definitions must be given for the auxiliary enzymes in the recommendations of standard determinations for enzyme activities.

Alanine Transaminase↗
PubMed1987

Macroenzyme as a cause of unexplained elevation of aspartate aminotransferase.

Aspartate aminotransferase (AST) can exist as a macroenzyme by forming a complex with an immunoglobulin. This immunoglobulin-complexed macromolecule can cause an elevation in serum AST activity, which may be detected on routine blood chemistry analysis and erroneously considered to indicate the presence of liver disease. Clinicians should be aware of this phenomenon so patients are not subjected to unnecessary procedures. In patients with unexplained AST elevation, liver and muscle disease can be biochemically excluded by the finding of normal serum levels of alanine aminotransferase and creatine kinase. The presence of macro-AST can be determined by exclusion chromatography, electrophoresis, and activation assays with pyridoxal 5-phosphate. The elevated AST values can persist for many years.

Aged↗
PubMed1997

Use of 1H-15N heteronuclear multiple-quantum coherence NMR spectroscopy to study the active site of aspartate aminotransferase.

Aspartate aminotransferase from Escherichia coli, an 88 kDa enzyme, was uniformly and selectively enriched with 15N and was studied by heteronuclear multiple-quantum coherence NMR spectroscopy in H2O. Good resolution was obtained for the downfield region (above 9.5 ppm chemical shift in the 1H dimension) for NH protons in the amide, indole, imidazole, and guanidinium group regions and several resonances were tentatively assigned. Two downfield resonances, at 12.6 and 11.36 ppm, appear to belong to oxygen- or sulfur-bound protons. The most downfield amide resonance at 11.78 ppm was assigned to the active site cysteine 192 whose peptide proton is 2.9 A away from the negatively charged carboxyl group of aspartate 199. Large downfield shifts (up to 1.15 ppm) of the indole NH resonance of the active site tryptophan 140 were observed upon binding of dicarboxylic inhibitors to the pyridoxal 5'-phosphate (PLP) form and of inorganic dianions to the pyridoxamine 5'-phosphate (PMP) form of the enzyme. We discuss these striking differences in the light of the available crystallographic data. Active sites of proteins, as well as specific inhibitory molecules, often contain negatively charged groups. These may be able to form hydrogen-bonds to NH groups and to shift the NH resonances downfield into a less crowded and therefore more readily observable region for many large proteins. Our approach, which makes use of both HMQC spectroscopy and NOE observations, should be widely applicable.

Aspartate Aminotransferases↗
PubMed1993

Comparative studies on thermophilicity and thermostability of aspartate aminotransferases.

Aspartate aminotransferase from Sulfolobus solfataricus (AspATSs) is an extremely thermophilic and thermostable enzyme. In order to investigate the structural features which underlie thermophilicity and thermostability, two isoforms of AspATSs differing by a single amino acid residue were compared. The first isoform is the naturally occurring enzyme, whereas the second is a genetically engineered mutant. Thermophilicity, short-term and long-term thermostability of the isoenzymes were independently evaluated and the influence of a cysteine residue on the three properties was assessed.

Aspartate Aminotransferases↗
PubMed1989

Purification and characterization of chicken brain cytosolic aspartate aminotransferase.

Aspartate aminotransferase from the cytosolic fraction of chicken brain was isolated with acceptable yield and high degree of purity. The enzyme appeared in multiple molecular forms: alpha, beta, gamma, and delta (alpha predominates), as detected by polyacrylamide gel electrophoresis with specific staining. These different forms of the enzyme were separated by DEAE-Sephacel chromatography, and showed different isoelectric points and maximal velocities values, whereas their molecular weight, optimum pH and Michaelis constants were very similar. Generation process studies suggest that minors subforms of the enzyme could be raised from alpha form by a mechanism in which the oxidation of particular amino acid groups are involved.

Animals↗
PubMed1997

Transient-state kinetics of the reaction of aspartate aminotransferase with aspartate at low pH reveals dual routes in the enzyme-substrate association process.

In aspartate aminotransferase, the coenzyme pyridoxal 5'-phosphate forms a Schiff base with the epsilon-amino group of Lys258. The pH dependency of the steady-state kinetics of the overall reaction had indirectly suggested that the Schiff-base-unprotonated form of the enzyme (EL) is the active species that binds the monoanionic form of aspartate (SH+), the predominant species of the substrate in solution. In order to obtain direct information on the association process, we carried out transient-phase kinetics of the first half-reaction of the enzyme with aspartate at various pH. The disappearance of EL (lambdamax = 358 nm) was fast and independent of pH, but the disappearance of ELH+ (Schiff-base-protonated form, lambdamax = 430 nm) was slow and dependent on pH. At pH values below 6.8 and low concentrations of aspartate, the results could be interpreted to indicate that EL reacts rapidly with SH+ to form the pyridoxamine 5'-phosphate form of the enzyme (EM), and the reaction of ELH+ proceeds via the route ELH+ right arrow over left arrow EL right arrow over left arrow EM, where the first step was found to be rate limiting from the pH jump/drop study of the enzyme. At higher pH values, the rate of disappearance of ELH+ became larger than expected from the above scheme. This deviation became apparent with increasing pH, and could be excellently explained if we consider that it is due to the reaction of ELH+ with the dianionic form of aspartate (S). Thus, the formation of the Michaelis complex of aspartate aminotransferase and aspartate can proceed via two routes; route A is the association of EL with SH+ to form EL.SH+, which converts intramolecularly to ELH+.S, and route B is the association of ELH+ with S to form ELH+.S directly. ELH+.S is the prerequisite structure for further processing of the substrate by the enzyme. The reactions of EM and oxo acids yielded almost exclusively EL and SH+, and therefore route B does not seem to play an essential role in the overall reactions of the enzyme. Route B, however, may be important in the reaction mechanisms of other pyridoxal 5'-phosphate enzymes which have only the ELH+ form.

Aspartate Aminotransferases↗
PubMed1993

Synthesis of 3-arsonoalanine and its action on aspartate aminotransferase and aspartate ammonia-lyase. Comparison with arsenical analogues of malate and fumarate.

DL-3-Arsonoalanine has been synthesized by the Strecker synthesis from the unstable compound arsonoacetaldehyde. It inactivates pig heart cytosolic aspartate aminotransferase and inhibits aspartate ammonia-lyase by competing with aspartate (Ki/Km 0.23). The fumarate analogue (E)-3-arsonoacrylic acid and the malate analogue (RS)-3-arsonolactate also inhibit fumarate hydratase, competing with fumarate (Ki/Km 1.8) and malate (Ki/Km 1.6) respectively. Attempted non-enzymic transamination of 3-arsonoalanine gave elimination of arsenite, in contrast with the transamination of 3-phosphonoalanine, which is either successful or leads to loss of phosphate.

Alanine↗