Syncatalytic sulfhydryl group modification in mitochondrial aspartate aminotransferase from chicken and pig heart

One sulfhydryl group of the mitochondrial isoenzyme of aspartate aminotransferase from both chicken and pig heart exhibits syncatalytic reactivity changes similar to those found previously in the cytosolic isoenzyme from pig heart (Birchmeier, W., Wilson, K.J., and Christen, P. (1973) J. Biol. Chem. $\text{248}$, 1751–1759). The reactivity of the only titratable sulfhydryl group toward 5,5′-dithiobis-(2-nitrobenzoate) is at a minimum in the free pyridoxal and pyridoxamine form of the enzyme and is increased by approximately one order of magnitude when covalent enzyme-substrate intermediates are formed. The modification of the sulfhydryl group does not affect enzymatic activity. This finding supports the earlier conclusion that the syncatalytic reactivity changes are not due to a direct participation of this group in the active site but rather to conformational adaptations of the enzyme-coenzyme-substrate compound occurring in the catalytic mechanism of aspartate aminotransferases.     

Inactivation of aspartate aminotransferase during transamination with chloropyruvate. Evidence against modification of Cys390 in cytosolic isoenzyme.

Both the cytosolic and mitochondrial isoenzyme of aspartate aminotransferase from pig heart were inactivated during transamination with chloropyruvate. Inactivation occurred with L-alanine as the amino group donor in the presence of potassium formate. When L-glutamate or L-aspartate was employed as the amino group donor in the transamination reaction with chloropyruvate, no inactivation occurred. This is in contrast to the case of inactivation by bromopyruvate (Okamoto, M. &; Morino, Y. (1973) J. Biol. Chem. $\text{248}$, 82–90) where these natural dicarboxylic amino acid substrates were effective in the transamination reaction leading to syncatalytic inactivation (Birchmeier, W. &; Christen, P. (1974) J. Biol. Chem. $\text{249}$, 6311–6315). The Cys₃₉₀ in the cytosolic isoenzyme which was modified in the syncatalytic inactivation was not modified under the present condition for inactivation with either chloropyruvate or bromopyruvate.     

Cysteine sulfinate transamination activity of aspartate aminotransferases.

Aspartate aminotransferases from pig heart cytosol and mitochondria, $\text{Escherichia coli}$ B and $\text{Pseudomonas striata}$ accepted L-cysteine sulfinate as a good substrate. The mitochondrial isoenzyme and the $\text{Escherichia}$ enzyme showed higher activity toward L-cysteine sulfinate than toward the natural substrates, L-glutamate and L-aspartate. The cytosolic isoenzyme catalyzed the L-cysteine sulfinate transamination at 50% the rate of L-glutamate transamination. The $\text{Pseudomonas}$ enzyme had the same reactivity toward the three substrates. Antisera against the two isoenzymes and the $\text{Escherichia}$ enzyme inactivated almost completely cysteine sulfinate transamination activity in the crude extracts of pig heart muscle and $\text{Escherichia coli}$ B, respectively. These results indicate that cysteine sulfinate transamination is catalyzed by aspartate aminotransferase in these cells.     

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.     

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.     

Aspartate transaminase from E. coli: amino acid sequences of the NH2-terminal 33 residues and chymotryptic pyridoxyl tetrapeptide.

The amino acid sequences of pyridoxal-binding tetrapeptide and the NH₂-terminal portion of aspartate transaminase from $\text{E.}$$\text{coli}$ B were analyzed and compared with those of the corresponding parts of the cytosolic and mitochondrial isozymes from pig heart. After borohydride reduction and chymotryptic digestion of the $\text{E.}$$\text{coli}$ enzyme, a pyridoxal-containing peptide was isolated, showing the sequence, Ser-Lys(Pxy)-Asn-Phe, identical with that of the cytosolic isozyme. The NH₂-terminal sequence was determined up to 33 residues with a liquid phase sequence analyzer. Nearly the same degree of homology was observed among the NH₂-terminal sequences of the three aspartate transaminases.     

Mitochondrial and cytosolic aspartate aminotransferase from chicken: Activity toward aromatic amino acids

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase from chicken heart accept as substrates L-phenylalanine, L-tyrosine and L-tryptophan. The specific activities of the mitochondrial isoenzyme toward these substrates are between 0.1 to 0.5% of that toward aspartate and two orders of magnitude higher than that toward alanine. The specific activities of the cytosolic isoenzyme toward the aromatic substrates are 10 to 70% of the respective values of the mitochondrial isoenzyme. The activities of both isoenzymes toward aromatic amino acids are increased two- to threefold by 1 M formate. Larger increases by formate were observed for the alanine aminotransferase activity of both isoenzymes whereas their aspartate aminotransferase activity was inhibited by formate. The opposite effects of formate on the activities toward the aromatic and aliphatic monocarboxylic substrates on the one hand and the dicarboxylic substrate on the other are consonant with the notion of formate occupying the binding site of the distal carboxylate group of the substrate (Morino Y., Osman A.M., and Okamoto M. (1974) J. Biol. Chem. $\text{249}$, 6684–6692). Apparently, in the ternary complex of aspartate aminotransferase with formate and aromatic amino acids, the aromatic rings of the latter bind to a site which does not overlap with the binding site for the distal carboxylate.     

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.     

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.     

The sequences of the coenzyme-binding peptide in the cytoplasmic and the mitochondrial aspartate aminotransferases from sheep liver.

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.     

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.     

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.     

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.     

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     

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)     

The covalent structure of mitochondrial aspartate aminotransferase from chicken. Identification of segments of the polypeptide chain invariant specifically in the mitochondrial isoenzyme

The primary structure of mitochondrial aspartate aminotransferase from chicken is reported. The enzyme is a dimer of identical subunits. Each subunit contains 401 amino acid residues; the calculated subunit molecular weight of the apoform is 44,866. The degree of sequence identity with the homologous cytosolic isoenzyme from chicken is 46%. A comparison of the primary structures of the mitochondrial and the cytosolic isoenzyme from pig and chicken shows that 40% of all residues are invariant. The degree of interspecies sequence identity both of the mitochondrial and the cytosolic isoenzyme from chicken and pig (86% and 83%, respectively) markedly exceeds that of the intraspecies identity between mitochondrial and cytosolic aspartate aminotransferase in chicken (46%) or in pig (48%). Based on these values, the duplication of the aspartate aminotransferase ancestral gene is estimated to have occurred approximately 1000 million years ago, i.e. at the time of the emergence of eukaryotic cells. By sequence comparison it is possible to identify amino acid residues and segments of the polypeptide chain that have been conserved specifically in the mitochondrial isoenzyme during phylogenetic evolution. These segments comprise about a third of the total polypeptide chain and appear to cluster in a certain surface region. The cluster carries an excess of positively charged residues which exceeds the overall charge difference between the cytosolic (pI approximately 6) and the mitochondrial isoenzyme (pI approximately 9).     

Isolation,crystallization and preliminary crystallographic data of aspartate aminotransferase from chicken heart mitochondria

The mitochondrial isoenzyme of aspartate aminotransferase (E.C. 2.6.1.1) has been isolated from chicken heart in an electrophoretically and immunologically homogeneous form. Large, well-diffracting single crystals of this enzyme, a dimeric molecule with a molecular weight of 90,000, have been grown by vapour phase diffusion against polyethylene glycol solutions. The crystals belong to space group P1. The unit cell, with the dimensions $\text{a = 55.6}\text{A}\text{?}\text{, 6 = 58.7}\text{A}\text{?}\text{, c = 76.0}\text{A}\text{?}$, α = 85.3 °, β = 109.2 °, γ = 115.6 °, contains a single dimer. The diffraction pattern extends to at least 2.1 Å resolution.     

Cloning and sequence analysis of a cdna encoding bovine cytosolic aspartate aminotransferase

• 1.1. Complementary DNA encoding cytosolic aspartate aminotransferase was isolated from an adult bovine heart library.
• 2.2. The amino add sequence deduced for the protein (412 amino acids) is extremely similar (> 94% identity) to that of porcine cytosolic aspartate aminotransferase but interesting differences were noticed comparing the position of cysteine residues.

     

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.     

Glutamate transport in pig heart mitochondria. Binding and structural properties of high-glutamate affinity proteolipid: Reconstitution studies

Previously, a proteolipid that can bind glutamate with high affinity has been isolated from pig heart mitochondrial membranes. A final affinity chromatography on γ-methylglutamate-albumin coreticulated on glass fiber was necessary. This procedure includes long dialysis steps which tend to denature the high-glutamate affinity proteolipid.Here is described a new method of isolation which avoids long dialysis steps and yields greater amounts of the high-glutamate affinity proteolipid.The binding of glutamate or aspartate on high-glutamate affinity proteolipid has been studied by gel filtration, by equilibrium dialysis or by a new procedure of rapid centrifugation based on the insolubility of high-glutamate affinity proteolipid in water. The latter method permits the detection of low and high affinity sites for glutamate with a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ 60 mM and 55 μM, respectively. Among a series of analogues, aspartate appeared to be the best competitor: $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 30 μ}\text{M}$ and two $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values, 0.37 mM (at high glutamate concentration) and 3.8 μM (at low glutamate concentration). High-glutamate affinity proteolipid binds 0.4 nmol of glutamate but only 0.1 nmol of aspartate per mg protein. The sites for glutamate and aspartate appear to be different but interdependent.In the presence of high-glutamate affinity proteolipid, externally added glutamate stimulated the efflux of aspartate from preloaded liposomes.High-glutamate affinity proteolipid contains cardiolipin, phosphatidyl choline and phosphatidyl ethanolamine the distribution of which is different from that of the inner membrane.The effects of various phospholipases, trypsin, and thiol reagents were studied on the binding of glutamate. High-glutamate affinity proteolipid binds 9 nmol $\text{N-}\text{ethylmaleimide}$ per mg protein but only 6.1 nmol in the presence of glutamate. The dissociation of high-glutamate affinity proteolipid caused by thiol reagents yielded a soluble protein fraction with higher affinity for glutamate.Electrophoresis and an immunological approach allowed the detection and titration of the glutamate dehydrogenase and aspartate aminotransferase present in high-glutamate affinity proteolipid in inhibited forms, the latter being 26-fold more concentrated than the former.