Conspicuous degree of homology between the mitochondrial aspartate aminotransferases from chicken and pig heart.

The sequence of 40 amino acid residues at the amino terminus of mitochondrial aspartate aminotransferase from chicken heart differs in only 2 positions from the sequence of mitochondrial aminotransferase of pig heart. Close structural similarity had been suggested by previous data on syncatalytic sulfhydryl modifications (Gehring H., and Christen P. (1975) Biochem. Biophys. Res. Commun. $\text{63}$, 441–447). The cytosolic aspartate aminotransferases from the same two species have now been found to differ considerably in the mode of their syncatalytic modifications. The data suggest that the cytosolic and mitochondrial aspartate aminotransferases might have evolved at different organelle-specific rates.     

Primary structure of aspartate aminotransferase from horse heart and comparison with that of other homotopic and heterotopic isoenzymes

Sulphydryl groups of mitochondrial aspartate aminotransferase from horse heart were titrated with 5,5'-dithiobis (2-nitrobenzoic acid). From analysis of peptic peptides, 378 amino acid residues (94.3% of the total) in the protein were identified. The results of amino acid sequence analysis are compared with those of cytosolic and mitochondrial aspartate aminotransferases from other sources.     

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. Correspondence to: C.J. Marshall     

The complete amino acid sequences of cytosolic and mitochondrial aspartate aminotransferases from horse heart,and inferences on evolution of the isoenzymes

Summary We report here the complete amino acid sequences of the cytosolic and mitochondrial aspartate aminotransferases from horse heart. The two sequences can be aligned so that 48.1% of the amino acid residues are identical. The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoenzymes from pig, chicken, rat, and human, and the enzyme fromEscherichia coli. The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly. Based on the rate of evolution of the mammalian isoenzymes, the geneduplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is estimated to have occurred at least 10⁹ years ago. The cytosolic and mitochondrial isoenzymes are equally related to the enzyme fromE. coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3×10⁹ years ago.     

Functional inhibition of cytosolic and mitochondrial aspartate aminotransferase by l-2-amino-4-methoxy-trans-3-butenoic acid in isolated rat hepatocytes and mitochondria

The transaminase inhibitor l-2-amino-4-methoxy-trans-3-butenoic acid (AMB) decreased aspartate aminotransferase activity by approximately two-thirds in isolated rat liver mitohondria incubated with succinate, ammonia, and ornithine. Aspartate production by the mitochondria was unaffected over the 30-min incubation period, indicating that mitochondrial aspartate aminotransferase activity is normally far in excess of that required for maximal rates of aspartate production. In rat hepatocytes incubated with lactate, ammonia, and ornithine the inhibition of both the cytosolic and mitochondrial isozymes of aspartate aminotransferase by AMB was partially blocked by the presence of ammonia and ornithine. When pyruvate was substituted for lactate as a carbon source with isolated hepatocytes, the presence of ammonia and ornithine blocked the inhibition by AMB of the mitochondrial but not the cytosolic isozyme of aspartate aminotransferase. Urea formation by cells incubated with lactate, ammonia, and ornithine was unaffected by AMB unless the cells were preincubated with the inhibitor prior to the addition of substrates. However, urea formation by cells incubated in the presence of pyruvate, ammonia, and ornithine was inhibited strongly by AMB even without preincubation. The results suggest that the stimulation of ureogenesis from ammonia and ornithine by pyruvate involves the cytosolic isozyme of aspartate aminotransferase. In contrast, the stimulation of ureogenesis elicited by lactate primarily involved mitochondrial aspartate aminotransferase.     

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.     

Structural and genetic relationships between cytosolic and mitochondrial isoenzymes

The most common type of genetic relationship between cytosolic and mitochondrial isoenzymes will probably be found to be divergent evolution from a common ancestral form. This is firmly established for the aspartate aminotransferases and less directly so in other cases. The two isoenzymes of aspartate aminotransferase have evolved at roughly equal rates at the level of total amino acid sequence but certain limited surface regions of the mitochondrial form have been much more highly conserved than corresponding regions in the cytosolic protein; these regions probably play a role in topogenesis of the mitochondrial isoenzyme. It is of interest that nearly all mitochondrial proteins are initially synthesised as precursors of molecular weight greater than the mature forms. In the case of aspartate aminotransferase, and possibly of other such isoenzymes, the N-terminus of the mature protein is nearly coincident with that of the cytosolic isoenzyme. Hence during evolution either the gene for the mitochondrial isoenzyme has gained an extra coding region for this N-terminal extension or, less likely, the structural gene for the cytosolic form has suffered a sizeable terminal deletion. Cytosolic and mitochondrial superoxide dismutases have not shared a common ancestral form as shown by the fact that their primary structures are completely unrelated. On the other hand, the mitochondrial and prokaryotic enzymes are clearly related. There is now, however, evidence to suggest that some prokaryotes possess a copper/zinc enzyme related to the eukaryotic cytosolic form. Hence the possibility arises that primitive prokaryotes possessed both proteins. The copper/zinc superoxide dismutase has been retained in the cytosol of eukaryotic cells and a few bacterial species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of aspartate aminotransferase activity in yeasts, and purification and characterization of mitochondrial and cytosolic isoenzymes from Rhodotorula minuta [corrected

The distribution of aspartate aminotransferase activity in yeasts was determined. The number of species of the enzyme in each yeast was determined by zymogram analysis. All the yeasts, except for the genus Saccharomyces, showed two or three activity bands on a zymogram. From among the strains, Rhodotorula minuta [corrected] and Torulopsis candida were selected for examination of the existence of yeast mitochondrial isoenzymes, because these strains showed two clear activity bands on the zymogram and contained a high amount of the enzyme. Only one aspartate aminotransferase was purified from T. candida: the component in the minor band on the zymogram was not an isoenzyme of aspartate aminotransferase. On the other hand, two aspartate aminotransferases were purified to homogeneity from R. minuta [corrected]. The components in the main and minor activity bands on the zymogram were identified as the mitochondrial and cytosolic isoenzymes, respectively, in a cell-fractionation experiment. The enzymatic properties of these isoenzymes were determined. The yeast mitochondrial isoenzyme resembled the animal mitochondrial isoenzymes in molecular weight (subunits and native form), absorption spectrum, and substrate specificity. The amino acid composition was closely similar to that of pig mitochondrial isoenzyme. Rabbit antibody against the yeast mitochondrial isoenzyme, however, did not form a precipitin band with the pig mitochondrial isoenzyme.     

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.     

Reactivity of sulphydryl groups of cytosolic and mitochondrial bovine aspartate aminotransferases

Summary Reactivity of sulphydryl groups of cytosolic and mitochondrial aspartate aminotransferases from ox heart has been studied. A total of 5 and 7 cysteine residues per monomer are present in cAATo and mAATo, respectively. In native conditions only a single sulphydryl group can be titrated by Nbs₂ while the catalytic activity remains unchanged, however in the mitochondrial isozyme the reactivity depends on the functional state of the enzyme. Reactivity toward NEM reveals the existence of a syncatalytic sulphydryl group in the cytosolic isozyme. Titration of cAATo with pMB at pH 8 and pH 5 confirms the existence of two exposed sulphydryl groups with a different reactivity. The results compared with those reported on the corresponding isozymes from pig and chicken heart show that syncatalytic sulphydryl groups are of general occurrence in these enzymes.     

Bilirubin inhibition of enzymes involved in the mitochondrial malate-aspartate shuttle

The effect of increasing bilirubin concentrations upon the catalytic activity of a series of dehydrogenases and aminotransferases was examined. The particular enzymes were chosen to examine the effect of bilirubin upon the activity of enzymes responsible for the indirect transfer of reducing equivalents across the inner mitochondrial membrane. Malate dehydrogenase was inhibited at very low concentrations of bilirubin and showed competitive inhibition with respect to coenzyme of 2 μM, while the cytosolic form of this enzyme exhibited a 15 μM inhibition constant. Cytosolic glycerol-3-phosphate dehydrogenase was not appreciably inhibited by bilirubin. Both the mitochondrial and cytosolic forms of aspartate aminotransferase showed moderate competitive bilirubin inhibition with respect to substrates with a K_i of 30 μM with respect to 2-oxoglutarate and a K_i of 80 μM with respect to aspartate. Preincubation studies indicated that inhibition was reversible for all enzymes examined. These results are interpreted in terms of the inhibition of the malate-aspartate shuttle by relatively low concentrations of bilirubin.     

Comparison of folding rates of homologous prokaryotic and eukaryotic proteins

The rate of polypeptide chain elongation is up to one order of magnitude faster in prokaryotic cells than in eukaryotes. Here we report that the rates of in vitro refolding of orthologous prokaryotic and eukaryotic proteins correlate with their differential rates of biosynthesis. The mitochondrial and cytosolic aspartate aminotransferases of chicken and aspartate aminotransferase of Escherichia coli show pairwise sequence identities of 41-48% and nearly identical three-dimensional structures. Nevertheless, the prokaryotic enzyme refolded 6 times faster (at 25 degrees C) than the eukaryotic isoenzymes after denaturation in 6 m guanidine hydrochloride. Prokaryotic malate dehydrogenase and lactate dehydrogenase also renatured faster than their orthologous eukaryotic counterparts, suggesting that evolutionary pressure has adapted the rate of folding to the rate of elongation of polypeptide chains.     

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.     

Comparison of coenzymatic activities of 2-nor-2-hydroxymethyl pyridoxal 5′-phosphate for mitochondrial and cytoplasmic aspartate aminotransferases

2-Nor-2-hydroxymethyl pyridoxal 5′-phosphate served as a somewhat superior coenzyme to pyridoxal 5′-phosphate for cytoplasmic and mitochondrial aspartate aminotransferases in the usual assay system containing Tris-HCl buffer. In the presence of phosphate anion, however, the affinities of the analog for both enzymes and the V values of the reactions mediated by the cytoplasmic and mitochondrial aspartate aminotransferases reconstituted with the analog were more markedly decreased than in the cases of the native coenzyme. Various Vitamin B₆ enzymes tested hitherto were classified into at least three groups according to their attitudes toward the coenzyme analog.     

Methionine Regeneration and Aspartate Aminotransferase in Parasitic Protozoa

Aspartate aminotransferases have been cloned and expressed from Crithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, and Plasmodium falciparum and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the P. falciparum enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem. 214:549-561, 1993), a feature shared by sequences discovered in P. vivax and P. berghei. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant P. falciparum aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in Trichomonas vaginalis and G. intestinalis homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with a K(i) of 27 microm.     

Irreversible inactivation of aspartate aminotransferases during transamination with L-propargylglycine

L-Propargylglycine serves as an amino acid substrate in the transamination reaction catalyzed by both cytosolic and mitochondrial aspartate aminotransferases from pig heart. Incubation of these isoenzymes with L-propargylglycine alone did not result in the inactivation of these enzymes. However, the presence of 2-oxoglutarate or pyruvate caused gradual irreversible inactivation of these isoenzymes. The inactivation was greatly accelerated by the presence of formate ion. Inactivation of both isoenzymes with L-[2-¹⁴C]propargylglycine resulted in stoichiometric incorporation of the radioactive molecule. Drastic changes in the absorption and circular dichroic spectra of the enzymes which took place during the inactivation also indicated that the modification by L-propargylglycine is restricted to the active site of these isoenzymes.     

Complete amino acid sequence of rat liver cytosolic alanine aminotransferase

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.     

Aspartate transaminase from cytosol of the chicken muscles: Its purification and various properties]

Homogeneous cytosolic aspartate aminotransferase was prepared from chicken muscle. The purification procedure involves heat and ammonium sulfate fractionation, chromatography on ion-exchage cellulose CM-52 and crystallization of the enzyme. A comparison of some properties of aspartate aminotransferases from chicken skeletal muscle and heart has been made. Both enzymes were found identical in terms of their electrophoretic mobility, isoelectric point, pyridoxal phosphate content and the amount of SH-groups.     

Selective determination of fish aspartate aminotransferase isoenzymes by their differential sensitivity to proteases

Various proteases (proteinase K, subtilisin, trypsin and chymotrypsin) were used to study the selective inactivation of the aspartate aminotransferase (EC 2.6.1.1) isoenzymes of grey mullet (Mugil auratus Risso; Osteichthyes). The cytosolic isoenzyme was significantly inactivated by proteinase K, subtilisin and chymotrypsin, while the mitochondrial isoenzyme was sensitive only to proteinase K and to high doses of trypsin. Further identification of the aspartate aminotransferase isoenzymes was based on their discrete sensitivity toward chymotrypsin. Chymotrypsin (1 mg/ml) successfully inhibited purified cytosolic aspartate aminotransferase as well as cytosolic isoenzyme from plasma, whereas the mitochondrial form persisted unaffected. Similar results were obtained when examining liver and red muscle homogenates. This method revealed that the increased total activity of aspartate aminotransferase in fish plasma with induced acute liver injury, was partially a result of the mitochondrial isoenzyme leakage from damaged tissue.