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PubMed1976

The anomalous kinetics of coupled aspartate aminotransferase and malate dehydrogenase. Evidence for compartmentation of oxaloacetate.

Cytoplasmic aspartate aminotransferase and malate dehydrogenase were purified from pig heart. Kinetic parameters were determined for the separate reaction catalysed by each enzyme and used to predict the course of the coupled reaction: (see article). Although a lag phase should have been easily seen, none was detected. The same coupled reaction was also carried out by using radioactive aspartate in the presence of unlabelled oxaloacetate. The reaction was quenched with HClO4 after 70 ms and the specific radioactivity of the malate produced in this system was found to be essentially the same as that of the original aspartate. These results show that oxaloacetate produced by the aspartate aminotransferase is converted into malate by malate dehydrogenase before it equilibrates with the pool of unlabelled oxaloacetate and are consistent with a proposal that the enzymes are associated in a complex. However, no physical evidence of the existence of a complex could be found. An alternative means of compartmentation of the intermediate as an unstable isomer is considered.

Animals↗

PubMed1977

Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro.

1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo.

Ammonium Chloride↗

PubMed1981

Inhibition by cycloserine of mitochondrial and cytosolic aspartate aminotransferase in isolated rat hepatocytes.

Isolated hepatocytes were incubated with L-cycloserine and then treated with digitonin so that mitochondrial and cytosolic fractions were obtained in 5 s. Mitochondrial and total cellular aspartate aminotransferases (EC 2.6.1.1) were inactivated in parallel. The enzyme was also inhibited in isolated mitochondria incubated with L-cycloserine. These results, in contrast with previous reports, indicate that cycloserine reacts equally with mitochondrial and cytosolic aspartate aminotransferases.

Animals↗

PubMed1983

Catalytic activity of non-cross-linked microcrystals of aspartate aminotransferase in poly(ethylene glycol).

The molar activity of crystalline mitochondrial aspartate aminotransferase is decreased to 10% of that of the enzyme in solution. The activity was measured in suspensions of non-cross-linked microcrystals (average dimensions 22 microns X 5 microns X 0.8 microns) in 30% (w/v) poly(ethylene glycol). Kinetic tests ruled out the possibility that diffusion of the substrate in the crystals is rate-limiting. The observed decrease in catalytic efficiency can be attributed exclusively to crystal-packing effects. A direct inhibition by poly(ethylene glycol) is excluded because poly(ethylene glycol), with average Mr 6000, cannot penetrate the liquid channels of the crystals, owing to its large Stokes radius. The crystals examined were triclinic and of the same habit as those used for high-resolution X-ray-crystallographic analysis [Ford, Eichele & Jansonius (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2559-2563]. The catalytic competence of crystalline aspartate aminotransferase confirms the relevance of the spatial model of this protein for the elucidation of its mechanism of action.

Alanine↗

PubMed1990

The amino acid sequence of cytosolic aspartate aminotransferase from human liver.

1. The cytosolic aspartate aminotransferase was purified from human liver. 2. The isoenzyme contains four cysteine residues, only one of which reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) in the absence of denaturing agents. 3. The amino acid sequence of the isoenzyme is reported, as determined from peptides produced by digestion with trypsin and with CNBr, and from sub-digestion of some of these peptides with Staphylococcus aureus V8 proteinase. 4. The isoenzyme shares 48% identity of amino acid sequence with the mitochondrial form from human heart. 5. Comparisons of the amino acid sequences of all known mammalian cytosolic aspartate aminotransferases and of the same set of mitochondrial isoenzymes are reported. The results indicate that the cytosolic isoenzymes have evolved at about 1.3 times the rate of the mitochondrial forms. 6. The time elapsed since the cytosolic and mitochondrial isoenzymes diverged from a common ancestral protein is estimated to be 860 x 10(6) years. 7. Experimental details and confirmatory data for the results presented here are given in a supplementary paper that has been deposited as a Supplementary Publication SUP 50158 (25 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.

Amino Acid Sequence↗

PubMed1990

Acinar zonation of cytosolic but not organelle-bound activities of phosphoenolpyruvate carboxykinase and aspartate aminotransferase in guinea-pig liver.

In human liver, unlike in rat liver, there is no apparent acinar heterogeneity of total cellular activity of phosphoenolpyruvate carboxykinase [Wimmer, Luttringer & Columbi (1990) Histochemistry 93, 409-415]. Since the intracellular compartmentation of phosphoenolpyruvate carbonxykinase differs in rat and human liver, we examined the acinar heterogeneity of cytosolic and organelle-bound activities of this enzyme in the guinea pig, which shows a more similar intracellular compartmentation of enzyme activity to human liver than does the rat. Cytosolic phosphoenolpyruvate carboxykinase activity was higher in periportal than in perivenous hepatocytes, whereas the organelle-bound activity was similar in the two cell populations. Aspartate aminotransferase and alanine aminotransferase activities showed a similar distribution to phosphoenolpyruvate carboxykinase, with a higher cytosolic activity in periportal than in perivenous hepatocytes but a similar organelle-bound activity in the two cell populations. Data on the acinar zonation of enzymes determined in whole cells or tissue should be interpreted cautiously if the enzyme activity is present in more than one subcellular compartment.

Alanine Transaminase↗

PubMed1991

The amino acid sequence of the aspartate aminotransferase from baker's yeast (Saccharomyces cerevisiae).

1. The single (cytosolic) aspartate aminotransferase was purified in high yield from baker's yeast (Saccharomyces cerevisiae). 2. Amino-acid-sequence analysis was carried out by digestion of the protein with trypsin and with CNBr; some of the peptides produced were further subdigested with Staphylococcus aureus V8 proteinase or with pepsin. Peptides were sequenced by the dansyl-Edman method and/or by automated gas-phase methods. The amino acid sequence obtained was complete except for a probable gap of two residues as indicated by comparison with the structures of counterpart proteins in other species. 3. The N-terminus of the enzyme is blocked. Fast-atom-bombardment m.s. was used to identify the blocking group as an acetyl one. 4. Alignment of the sequence of the enzyme with those of vertebrate cytosolic and mitochondrial aspartate aminotransferases and with the enzyme from Escherichia coli showed that about 25% of residues are conserved between these distantly related forms. 5. Experimental details and confirmatory data for the results presented here are given in a Supplementary Publication (SUP 50164, 25 pages) that has been deposited at the British Library Document Supply Centre, Boston Spa. Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

Amino Acid Sequence↗

PubMed1991

Purification and characterization of aspartate aminotransferase from the halophile archaebacterium Haloferax mediterranei.

Aspartate aminotransferase from the archaebacterium Haloferax mediterranei was purified and found to be homogeneous. An average Mr of 66,000 was estimated. The native halophilic transaminase exhibited no maximum absorption at 410 nm, which indicates that the apo form is obtained by our purification procedure, and the molar absorption coefficient at 275 nm in 3.5 M-KCl (pH 7.8) was found to be 78.34 mM-1.cm-1. Plots of titration data show that 1 mol of halophilic aspartate aminotransferase binds 2 mol of pyridoxal 5'-phosphate. The halophilic transaminase behaved as a dimer with two similar subunits and had a maximum activity in the pH range 7.6-7.9 and at 65 degrees C in 3.5 M-KCl. By differential scanning calorimetry, the denaturation temperature of the halophilic holo- and apo-transaminase was determined to be 78.5 and 68.0 degrees C respectively at 3.3 M-KCl (pH 7.8). At low salt concentration the halophilic transaminase was inactivated, following first-order kinetics. The Km values for 2-oxoglutarate and L-aspartate, in 3 M-KCl (pH 7.8), were 0.75 mM and 12.6 mM respectively.

Amino Acids↗

PubMed1982

Multiple molecular forms of human heart cytoplasmic aspartate aminotransferase.

1. Cytoplasmic aspartate aminotransferase was isolated and purified from human heart with a final specific activity of 236 units/mg of protein. 2. Three distinct peaks of aspartate aminotransferase activity were detected by isoelectric focusing with isoelectric points of 5.46, 5.60 and 5.71. Two minor subforms were also noted as shoulder patterns with pI 5.2 and 5.8. 3. These electrophoretic characteristics are similar to previous findings of multiple molecular forms detected in human liver and erythrocytes.

Aspartate Aminotransferases↗

PubMed1995

Aspartate aminotransferase and glutamate dehydrogenase activities in the squid giant nerve.

The present study sought to investigate the presence and distribution of some enzymatic activities involved in the metabolism of glutamate in the giant nerve fiber of the tropical squid Sepioteuthis sepioidea. Specific activities of aspartate aminotransferase and glutamate dehydrogenase were evaluated in homogenates of the isolated giant fiber, extruded axoplasm, and axoplasm-free giant nerve fiber sheaths. The activities of both enzymes were present in the tissue. The specific activity of aspartate aminotransferase was similar in axoplasm and sheaths. However, the specific activity of glutamate dehydrogenase was an order of magnitude higher in the sheaths. This finding is discussed in the framework of the hypothesis that proposes that a differential distribution of the enzymes of the glutamatergic system between the axonal and neuroglial compartments forms part of a system of communication between these cells whose neuronal signal may be glutamate.

Animals↗

PubMedDate not supplied

The persistence and significance of elevated alanine aminotransferase levels in blood donors.

The level of alanine aminotransferase (ALT) in blood donors has been related to the frequency of posttransfusion hepatitis in recipients. Sixty-seven donors with elevated ALT levels were evaluated to define the duration and significance of the elevation. The ALT level remained elevated in 41 donors (61%) for a mean interval of 9 months. The ALT level was greater than the aspartate aminotransferase in all of the donors. Alcohol intake did not correlate with ALT level. Donors with persistently elevated ALT levels had a significantly higher mean percent ideal body weight (128 +/- 3.9) than donors whose ALT level became normal (116 +/- 3.1). Nine donors with elevated ALT levels for at least 6 months had needle biopsies of the liver. Seven had prominent fatty vacuolization of hepatocytes without evidence of alcoholic hepatitis. One biopsy demonstrated chronic persistent hepatitis. No other cause for the elevated ALT levels could be identified. An overweight male donor with an isolated ALT elevation may need no further investigation unless clinical evaluation suggests a source of liver injury.

Adolescent↗

PubMed1981

Effect of L-tryptophan injection in rats on some enzymes of amino acid metabolism in liver. I. In vitro studies of the effect of L-tryptophan and its metabolites on the extramitochondrial L-alanine: 2-ketoglutaric aminotransferase.

Fed and fasted rats were injected with L-tryptophan (12.5 mg/100 g body weight) and the specific activities of L-glutamic: NAD oxidoreductase (deaminating) (EC 1.4.1.2) (GDH), L-aspartic-2-ketoglutaric aminotransferase (EC 2.6.1.1) (GOT) and L-alanine-2-ketoglutaric aminotransferase (EC 2.6.1.2) (GPT) from hepatic mitochondria and cytosol were compared. L-tryptophan results in a decrease of mitochondrial GDH activity by 22% and of cytosolic GPT and GOT by 42% and 38% respectively in the liver of fasted rats. Xanthurenate is a potent inhibitor of purified extramitochondrial GPT, whereas anthranilate and quinolinate are less potent inhibitors. L-tryptophan, 5-OH-tryptophan and indole exert a slight inhibition. Kynurenine, 5-OH-tryptamine, tryptamine, picolinic acid, nicotinic acid and indoloacetic acid do not show any inhibition of GPT. It is suggested that L-tryptophan injection inhibits extramitochondrial GPT by its transformation to xanthurenate and anthranilate.

Alanine Transaminase↗

PubMed1978

Syncatalytic conformational changes in aspartate aminotransferase determined by hydrogen-deuterium exchange.

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.

Animals↗

PubMed1980

Three-dimensional structure of a pyridoxal-phosphate-dependent enzyme, mitochondrial aspartate aminotransferase.

X-ray diffraction studies to 2.8-A resolution have yielded the three-dimensional structure of mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), an isologous alpha 2 dimer (Mr = 2 x 45,000). The subunits are rich in secondary structure and contain two domains, one of which anchors the coenzyme, pyridoxal 5'-phosphate. Each active site lies between the subunits and is composed of residues from both of them.

Animals↗

PubMed1983

Crystalline aspartate aminotransferase: lattice-induced functional asymmetry of the two subunits.

The enzymic activity of crystalline mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) was determined in suspensions of noncrosslinked microcrystals in 30% (wt/vol) polyethylene glycol. The crystals (average dimensions, 22 x 5 x 0.8 micron) were small enough to preclude diffusional rate limitation. They had the same habit as the triclinic crystals used for the determination of the spatial structure of the enzyme by x-ray crystallographic analysis [Ford, G. C., Eichele, G., and Jansonius, J. N. (1980) Proc. Natl. Acad. Sci. USA 77, 2559-2563]. Determination of the Michaelis-Menten parameters showed that the packing of the enzyme dimer into the crystal lattice not only decreases its activity but also induces a functional nonequivalence of the two subunits that behave identically in solution. The crystalline enzyme possesses a high-affinity subunit with Km values similar to those of the enzyme in solution (K'm = 0.5 mM for aspartate and 1.2 mM for 2-oxoglutarate) and a low-affinity subunit (K'm = 5.5 mM and 14.5 mM, respectively). The catalytic activity of the high-affinity subunit is 3% and that of the low-affinity subunit is 15% of the activity of the enzyme in solution. The functional asymmetry of the crystalline enzyme dimer could also be demonstrated by selective mechanism-based modification of either type of active sites. In view of the apparently identical conformation of the two subunits in the crystalline enzyme, its decreased catalytic efficiency and its functional asymmetry likely are due to constraints exerted by the crystal lattice on the conformational adaptability of the two subunits. In triclinic crystals the two subunits of the enzyme dimer have dissimilar lattice contacts.

Animals↗

PubMed1985

Cloning and sequence analysis of a cDNA encoding porcine mitochondrial aspartate aminotransferase precursor.

The primary structure of pig mitochondrial aspartate aminotransferase (mAspATase; L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) precursor was deduced from its cDNA sequence. A library of cDNA clones was constructed from pig liver poly(A)+ RNA by applying the vector/primer method of Okayama and Berg [Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, 161-170]. The library was screened for pig mAspATase sequences by using a mixture of eight oligodeoxyribonucleotides as a probe. The sequences of the probe were deduced from the known amino acid sequence of pig mAspATase residues 196-201. Two recombinant plasmids containing inserts of about 2500 and 2600 base pairs were selected for sequence analysis. The amino acid sequence predicted from the cDNA sequence shows that the pig mAspATase precursor consists of the mature enzyme of 401 amino acid residues and an amino-terminal segment of 29 amino acid residues called the "presequence" that contains four basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The sequence of this 29-amino acid mAspATase precursor segment was compared with the presequences of other mitochondrial enzymes.

Amino Acid Sequence↗

PubMed1998

Thermodynamics and molecular simulation analysis of hydrophobic substrate recognition by aminotransferases.

Aromatic amino acid aminotransferase (AroAT) and aspartate aminotransferase (AspAT) are known as dual-substrate enzymes, which can bind acidic and hydrophobic substrates in the same pocket (Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and Kuramitsu, S. (1997) J. Biochem. (Tokyo) 122, 55-63). In order to elucidate the mechanism of hydrophobic substrate recognition, kinetic and thermodynamic analyses using substrates with different hydrophobicities were performed. They revealed that 1) amino acid substrate specificity (kmax/Kd) depended on the affinity for the substrate (1/Kd) and 2) binding of the hydrophobic side chain was enthalpy-driven, suggesting that van der Waals interactions between the substrate-binding pocket and hydrophobic substrate predominated. Three-dimensional structures of AspAT and AroAT bound to alpha-aminoheptanoic acid were built using the homology modeling method. A molecular dynamic simulation study suggested that the outward-facing position of the Arg292 side chain was the preferred state to a greater extent in AroAT than AspAT, which would make the hydrophobic substrate bound state of the former more stable. Furthermore, AroAT appeared to have a more flexible conformation than AspAT. Such flexibility would be expected to reduce the energetic cost of conformational rearrangement induced by substrate binding. These two mechanisms (positional preference of Arg and flexible conformation) may account for the high activity of AroAT toward hydrophobic substrates.

Aspartate Aminotransferases↗

PubMed1999

Conversion of aspartate aminotransferase into an L-aspartate beta-decarboxylase by a triple active-site mutation.

The conjoint substitution of three active-site residues in aspartate aminotransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ratio of L-aspartate beta-decarboxylase activity to transaminase activity >25 million-fold. This result was achieved by combining an arginine shift mutation (Y225R/R386A) with a conservative substitution of a substrate-binding residue (R292K). In the wild-type enzyme, Arg(386) interacts with the alpha-carboxylate group of the substrate and is one of the four residues that are invariant in all aminotransferases; Tyr(225) is in its vicinity, forming a hydrogen bond with O-3' of the cofactor; and Arg(292) interacts with the distal carboxylate group of the substrate. In the triple-mutant enzyme, k(cat)' for beta-decarboxylation of L-aspartate was 0.08 s(-1), whereas k(cat)' for transamination was decreased to 0.01 s(-1). AspAT was thus converted into an L-aspartate beta-decarboxylase that catalyzes transamination as a side reaction. The major pathway of beta-decarboxylation directly produces L-alanine without intermediary formation of pyruvate. The various single- or double-mutant AspATs corresponding to the triple-mutant enzyme showed, with the exception of AspAT Y225R/R386A, no measurable or only very low beta-decarboxylase activity. The arginine shift mutation Y225R/R386A elicits beta-decarboxylase activity, whereas the R292K substitution suppresses transaminase activity. The reaction specificity of the triple-mutant enzyme is thus achieved in the same way as that of wild-type pyridoxal 5'-phosphate-dependent enzymes in general and possibly of many other enzymes, i.e. by accelerating the specific reaction and suppressing potential side reactions.

Aspartate Aminotransferases↗