Phenylalanine- and tyrosine-auxotrophic mutants of Saccharomyces cerevisiae impaired in transamination

This paper reports the first isolation of Saccharomyces cerevisiae mutants lacking aromatic aminotransferase I activity (aro8), and of aro8 aro9 double mutants which are auxotrophic for both phenylalanine and tyrosine, because the second mutation, aro9, affects aromatic aminotransferase II. Neither of the single mutants displays any nutritional requirement on minimal ammonia medium. In vitro, aromatic aminotransferase I is active not only with the aromatic amino acids, but also with methionine, α-aminoadipate, and leucine when phenylpyruvate is the amino acceptor, and in the reverse reactions with their oxo-acid analogues and phenylalanine as the amino donor. Its contribution amounts to half of the glutamate:2-oxoadipate activity detected in cell-free extracts and the enzyme might be identical to one of the two known α-aminoadipate aminotransferases. Aromatic aminotransferase I has properties of a general aminotransferase which, like several aminotransferases of Escherichia coli, may be able to play a role in several otherwise unrelated metabolic pathways. Aromatic aminotransferase II also has a broader substrate specificity than initially described. In particular, it is responsible for all the measured kynurenine aminotransferase activity. Mutants lacking this activity grow very slowly on kynurenine medium. Received: 21 October 1996 / Accepted: 23 September 1997     

Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily

The ARO8 and ARO9 genes of Saccharomyces cerevisiae were isolated by complementation of the phenylalanine/tyrosine auxotrophy of an aro8 aro9 double-mutant strain that is defective in aromatic aminotransferases I (aro8) and II (aro9). The genes were sequenced, and deletion mutants were constructed and analysed. The expression of ARO8 and ARO9 was studied. The deduced amino acid sequences of Aro8p and Aro9p suggest that the former is a 500-residue, 56168-Da polypeptide and the latter a 513-residue, 58516-Da polypeptide. They correspond, respectively, to Ygl202p and Yhr137p, two putative proteins of unknown function revealed by systematic sequencing of the yeast genome. We show that aromatic aminotransferases I and II are homologous proteins, members of aminotransferase subgroup I, and, together with three other proteins, they constitute within the subgroup a new subfamily of enzymes specialised for aromatic amino acid and α-aminoadipate transamination. ARO8 expression is subject to the general control of amino acid biosynthesis. ARO9 expression is induced when aromatic amino acids are present in the growth medium and also in aro8 mutants grown on minimal ammonia medium. An autonomously replicating sequence (ARS) element is located between the ARO8 gene and YGL201c which encodes a protein of the minichromosome maintenance family.     

Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily

The ARO8 and ARO9 genes of Saccharomyces cerevisiae were isolated by complementation of the phenylalanine/tyrosine auxotrophy of an aro8 aro9 double-mutant strain that is defective in aromatic aminotransferases I (aro8) and II (aro9). The genes were sequenced, and deletion mutants were constructed and analysed. The expression of ARO8 and ARO9 was studied. The deduced amino acid sequences of Aro8p and Aro9p suggest that the former is a 500-residue, 56168-Da polypeptide and the latter a 513-residue, 58516-Da polypeptide. They correspond, respectively, to Ygl202p and Yhr137p, two putative proteins of unknown function revealed by systematic sequencing of the yeast genome. We show that aromatic aminotransferases I and II are homologous proteins, members of aminotransferase subgroup I, and, together with three other proteins, they constitute within the subgroup a new subfamily of enzymes specialised for aromatic amino acid and α-aminoadipate transamination. ARO8 expression is subject to the general control of amino acid biosynthesis. ARO9 expression is induced when aromatic amino acids are present in the growth medium and also in aro8 mutants grown on minimal ammonia medium. An autonomously replicating sequence (ARS) element is located between the ARO8 gene and YGL201c which encodes a protein of the minichromosome maintenance family. Received: 18 June 1997 / Accepted: 23 September 1997     

Tryptophan degradation in Saccharomyces cerevisiae: Characterization of two aromatic aminotransferases

Tryptophan was found to be degraded in Saccharomyces cerevisiae mainly to tryptophol. Upon chromatography on DEAE-cellulose two aminotransferases were identified: Aromatic aminotransferase I was constitutively synthesized and was active in vitro with tryptophan, phenylalanine or tyrosine as amino donors and pyruvate, phenylpyruvate or 2-oxoglutarate as amino acceptors. The enzyme was six times less active with and had a twenty times lower affinity for tryptophan (K _m=6 mM) than phenylalanine or tyrosine. It was postulated thus that aromatic aminotransferase I is involved in vivo in the last step of tyrosine and phenylalanine biosynthesis. Aromatic aminotransferase II was inducible with tryptophan but also with the other two aromatic amino acids either alone or in combinations. With tryptophan as amino donor the enzyme was most active with phenylpyruvate and not active with 2-oxoglutarate as amino acceptor; its affinity for tryptophan was similar as for the other aromatic amino acids (K _m=0.2–0.4 mM). Aromatic aminotransferase II was postulated to be involved in vivo mainly in the degradation of tryptophan, but may play also a role in the degradation of the other aromatic amino acids.A mutant strain defective in the aromatic aminotransferase II (aat2) was isolated and its influence on tryptophan accumulation and pool was studied. In combination with mutations trp2 ^fbr, aro7 and cdr1-1, mutation aat2 led to a threefold increase of the tryptophan pool as compared to a strain with an intact aromatic aminotransferase II.     

Diversity of amino acid converting enzymes in wild lactic acid bacteria

A total of 156 lactic acid bacteria isolates belonging to the genera Lactococcus, Lactobacillus and Leuconostoc were analysed for the amino acid converting enzymes aminotransferases, glutamate dehydrogenase, and α-ketoisovalerate decarboxylase. All isolates showed aminotransferase activity towards phenylalanine (substrate for the aromatic aminotransferase AraT) and isoleucine (substrate for the branched-chain aminotransferase BcaT). Although there was a high variability inter- and intra-species, the lactococcal strains showed the highest values for both aminotransferase activities. Moreover, α-ketoisovalerate decarboxylase (Kivd) activity was only found in lactococcal isolates, although at low relative numbers (16%). On the other hand, glutamate dehydrogenase (Gdh) activity values were highest in facultative heterofermentative lactobacilli (FHL) and the activity was found at high relative numbers (50%) in leuconostocs. Results showed a high variability in amino acid convertase activities within the wild LAB isolates assayed, therefore the utilisation in the dairy industry of new strains with high flavour-forming abilities could be a powerful tool to enhance cheese aroma development.     

Influence of ring- and side-chain substituents on the transamination of aromatic amino acids by two multispecific aspartate-aromatic aminotransferase isozymes purified from bushbean shoots

The breadth of substrate specificity shown by the multispecific aspartate-aromatic aminotransferase of bushbean (Phaseolus vulgaris) has been investigated by testing the ability of two cytosolic isozymes (I and II), purified from shoot tissue, to catalyse transamination reactions between a range of ring- and sidechain-substituted aromatic amino acids and 2-oxoglutarate. Ring-substituted phenylalanines were the most reactive substrates whereas ring-substitution in tyrosine or tryptophan resulted in transamination rates lower than those observed with the parent amino acids. All side chain-substituted analogues were found to be totally inactive. The highest activity shown by any ring-substituted phenylalanine was observed with the 4-amino- compound, followed closely by the 4-hydroxy- and 4-halogen-compounds. In contrast, 4-nitrophenylalanine was completely inactive. These trends were consistent for both isozymes I and II, but only isozyme II showed greatly enhanced activity over that found with the parent amino acid when certain ring-substituted analogues were tested. The varying capacity of the bushbean isozymes to utilize the present range of substituted amino acids is compared with previous reports on the substrate specificity shown by aspartate and aromatic aminotransferases isolated from mammalian and microbial systems. A model for the mechanism of activation observed with bushbean isozyme II in the presence of certain 4-substituted aromatic amino acids is proposed, based on current understanding of the nature of the active site of animal aspartate aminotransferases.     

Molecular analysis of the role of two aromatic aminotransferases and a broad-specificity aspartate aminotransferase in the aromatic amino acid metabolism of Pyrococcus furiosus

The genes encoding aromatic aminotransferase II (AroAT II) and aspartate aminotransferase (AspAT) from Pyrococcus furiosus have been identified, expressed in Escherichia coli and the recombinant proteins characterized. The AroAT II enzyme was specific for the transamination reaction of the aromatic amino acids, and uses a-ketoglutarate as the amino acceptor. Like the previously characterized AroAT I, AroAT II has highest efficiency for phenylalanine (k(cat)/Km = 923 s(-1) mM(-1)). Northern blot analyses revealed that AroAT I was mainly expressed when tryptone was the primary carbon and energy source. Although the expression was significantly lower, a similar trend was observed for AroAT II. These observations suggest that both AroATs are involved in amino acid degradation. Although AspAT exhibited highest activity with aspartate and alpha-ketoglutarate (k(cat) approximately 105 s(-1)), it also showed significant activity with alanine, glutamate and the aromatic amino acids. With aspartate as the amino donor, AspAT catalyzed the amination of alpha-ketoglutarate, pyruvate and phenyl-pyruvate. No activity was detected with either branched-chain amino acids or alpha-keto acids. The AspAT gene (aspC) was expressed as a polycistronic message as part of the aro operon, with expression observed only when the aromatic amino acids were absent from the growth medium, indicating a role in the biosynthesis of the aromatic amino acids.     

Nonidentity of the Aspartate and the Aromatic Aminotransferase Components of Transaminase A in Escherichia coli

Tyrosine, added to the growth medium of a strain of Escherichia coli K-12 lacking transaminase B, repressed the tyrosine, phenylalanine, and tryptophan aminotransferase activities while leaving the aspartate aminotransferase activity unchanged. This suggested that the aspartate and the aromatic aminotransferase activities, previously believed to reside in the same protein, viz. transaminase A, are actually nonidentical. Further experiments showed that, upon incubation at 55 C, the aspartate aminotransferase of crude extracts was almost completely stable, whereas the tyrosine and phenylalanine activities were rapidly inactivated. Apoenzyme formation was faster, and apoenzyme degradation proceeded more slowly with aspartate aminotransferase than with tyrosine aminotransferase. Electrophoresis in polyacrylamide gels separated the aminotransferases. A more rapidly moving band contained tyrosine, phenylalanine, and tryptophan aminotransferases, and a slower band contained aspartate aminotransferase. A mutant of E. coli K-12 with low levels of aspartate aminotransferase exhibited unchanged levels of tyrosine aminotransferase. Thus, transaminase A appears to be made up of at least two proteins: one of broad specificity whose synthesis is repressed by tyrosine and another, specific for aspartate, which is not subject to repression by amino acids. The apparent molecular weights of both the aspartate and the aromatic aminotransferases, determined by gel filtration, were about 100,000.     

Purification and properties of two aromatic aminotransferases in Bacillus subtilis.

Two enzymes which transaminate tyrosine and phenylalanine in Bacillus subtilis were each purified over 200-fold and partially characterized. One of the enzymes, termed histidinol phosphate aminotransferase, is also active with imidazole acetyl phosphate as the amino group recipient. Previous studies have shown that mutants lacking this enzyme require histidine for growth. Mutants in the other enzyme termed aromatic aminotransferase are prototrophs. Neither enzyme is active on any other substrate involved in amino acid synthesis. The two enzymes can be distinguished by a number of criteria. Gel filtration analysis indicate the aromatic and histidinol phosphate aminotransferases have molecular weights of 63,500 and 33,000, respectively. Histidinol phosphate aminotransferase is heat-sensitive, whereas aromatic aminotransferase is relatively heat-stable, particularly in the presence of alpha-ketoglutarate. Both enzymes display typical Michaelis-Menten kinetics in their rates of reaction. The two enzymes have similar pH optima and employ a ping-pong mechanism of action. The Km values for various substrates suggest that histidinol phosphate aminotransferase is the predominant enzyme responsible for the transamaination reactions in the synthesis of tyrosine and phenylalanine. This enzyme has a 4-fold higher affinity for tyrosine and phenylalanine than does the aromatic aminotransferase. Competitive substrate inhibition was observed between tyrosine, phenylalanine, and histidinol phosphate for histidinol phosphate aminotransferase. The significance of the fact that an enzyme of histidine synthesis plays an important role in aromatic amino acid synthesis is discussed.     

Three Different Classes of Aminotransferases Evolved Prephenate Aminotransferase Functionality in Arogenate-competent Microorganisms

The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1β in tested α- and β-proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the β-proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.     

TRANSAMINATION OF AMINO ACIDS IN HOMOGENATES OF RAT BRAIN

Abstract— The aminotransferase activity of homogenates of brains from adult and neonatal rats has been investigated. Aminotransferase activity was demonstrated wtih 15 of 22 amino acids incubated with seven keto acids. The basic amino acids exhibited little or no activity.

• 1 The greatest activity was obtained when glutamate or aspartate was incubated with α-ketoglutarate or oxaloacetate. Significant activity was also observed when the neutral aliphatic and aromatic amino acids were incubated with these two keto acids.
• 2 Activity with pyruvate was obtained principally upon incubation with glutamate and alanine. Most of the other amino acids that underwent transamination with α-ketoglutarate also did so with pyruvate, although at a lower rate.
• 3 When phenylpyruvate was added to the medium, glutamate, phenylalanine and tyrosine transaminated most actively.
• 4 Incubations with 11 amino acids and glyoxylic acid demonstrated aminotransferase activity, with glutamate and ornithine being the most active substrates.
• 5 α-Ketoisocaproate and α-ketoisovalerate accepted amino groups primarily from the branched-chain amino acids. Except for glutamate, activity with other amino acids was low or not detectable.
• 6 A comparison of aminotransferase activity in the newborn brain with that in the adult brain showed that the greatest change in activity occurred for glutamate with pyruvate or for alanine with α-ketoglutarate, these activities increasing about 10-fold from birth to adulthood; during this time activities with most other amino acids increased two- to threefold. Amino transfers from the branched-chain amino acids showed no increase with maturation, and some reactions, such as that with methionine and a number of keto acids, decreased from birth to adulthood.
• 7 Our results correspond in general to previous studies of aminotransferase activity in brain and in liver. However, our study also indicates a possible second aminotransferase acting on the branched-chain amino acids, the presence of aminotransferase activity for methionine and asparagine, and relatively high aminotransferase activity for glutamine or ornithine when incubated with glyoxylic acid rather than other keto acids. Moreover, phenylpyruvate and glyoxylate are active in amino transfers and may serve as substrates for a number of aminotransferases.

     

Kynurenine aminotransferase and alpha-aminoadipate aminotransferase: III. Evidence for identity with halogenated tyrosine aminotransferase.

A nearly homogeneous preparation of α-aminoadipate (kynurenine) aminotransferase exhibited substantial activity with 3,5-diiodo-L-tyrosine, a major substrate for halogenated tyrosine aminotransferase. The new activity was found, according to heat inactivation and several inhibition studies, not to be attributable to contamination. Many of the properties previously reported for the two enzymes are identical or very similar. This paper lists these similarities and reports our observations of additional similarities of these activities in the supernatant and mitochondrial fractions of both rat kidney and liver. The properties of the purified enzyme and the noted similarities suggest that α-aminoadipate aminotransferase, kynurenine aminotransferase, and halogenated tyrosine aminotransferase activities are associated with the same protein. These activities are discussed in terms of a possible role in thyroid hormone metabolism.     

tyrB-2 and phhC genes of Pseudomonas putida encode aromatic amino acid aminotransferase isozymes: evidence at the protein level

Two Pseudomonas putida aminotransferases (ArAT I and ArAT II) that exhibit activity toward l-tryptophan were purified 104- and 395-fold using a six-stage purification procedure involving ammonium sulfate fractionation and chromatographic separation on phenyl-Sepharose, Sephadex G-100 superfine, DEAE-cellulose and Protein-Pack Q8 HR columns. Mass spectrometry analysis resulted in the identification of 27 and 20 % of the total ArAT I and ArAT II amino acid sequences. In addition, N-terminal sequence fragments of ArAT I and ArAT II were determined using the Edman degradation method. Based on the analyses performed, the studied proteins were identified as products of the tyrB-2 and phhC genes, and the presence of these genes in the investigated bacterial strain was confirmed using molecular biology methods. Extensive analysis of the substrate specificities of ArAT I and ArAT II revealed that both enzymes most efficiently catalyzed reactions involving aromatic amino acids and 2-oxoacids followed by dicarboxylic compounds. The best substrates for ArAT I and ArAT II were l-phenylalanine and phenylpyruvate. Based on these results, the studied proteins were classified as aromatic amino acid aminotransferase isozymes.     

Identity of kynurenine: pyruvate aminotransferase with histidine: pyruvate aminotransferase.

Kynurenine pyruvate aminotransferase was purified from rat kidney. The purified enzyme had an isoelectric point of pH 5.2 and a pH optimum of 9.3. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors. L-Amino acids were effective in the following order of activity: histidine greather than phenylalanine greater than kynurenine greater than tyrosine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values were about 0.63 mM, 1.4 mM and 0.09 mM for histidine, kynurenine and phenylalanine, respectively. Km values for pyruvate were 5.5 mM with histidine as amino donor, 1.3 mM with kynurenine and 8.5 mM with phenylalanine. Kynurenine pyruvate aminotransferase activity of the enzyme was inhibited by the addition of histidine or phenylalanine. The molecular weights determined by gel filtration and sucrose density gradient centrifugation were approximately 76000 and 79000, respectively. On the basis of purification ratio, substrate specificity, inhibition by common substrates, subcellular distribution, isoelectric focusing and polyacrylamide-gel electrophoresis, it is suggested that kynurenine pyruvate aminotransferase is identical with histidine pyruvate aminotransferase and also with phenylalanine pyruvate aminotransferase. The physiological significance of the enzyme is discussed.     

Crystal structures of glutamine:phenylpyruvate aminotransferase from Thermus thermophilus HB8: induced fit and substrate recognition

The following three-dimensional structures of three forms of glutamine:phenylpyruvate aminotransferase from Thermus thermophilus HB8 have been determined and represent the first x-ray analysis of the enzyme: the unliganded pyridoxal 5'-phosphate form at 1.9 A resolution and two complexes with 3-phenylpropionate and alpha-keto-gamma-methylthiobutyrate at 2.35 and 2.6 A resolution, respectively. The enzyme shows high activity toward phenylalanine, tyrosine, tryptophan, kynurenine, methionine, and glutamine. The enzyme is a homodimer, and each subunit is divided into an N-terminal arm and small and large domains. Based on its folding, the enzyme belongs to fold type I, aminotransferase subclass Ib. The subclass I aminotransferases whose structures have so far been determined exhibit a large movement of the small domain region upon binding of a substrate. Similarly, the glutamine:phenylpyruvate aminotransferase undergoes a large movement in part of the small domain to close the active site. The active-site pocket has a shape and size suitable to enclose the side chain of an aromatic amino acid or that of methionine. The inner side of the pocket is mostly hydrophobic, but also has polar sites. The kynurenine complex generated by computer modeling fits the pocket of the enzyme and its hydrophilic groups interact with the polar sites of the pocket.     

CEREBRAL AROMATIC AMINOTRANSFERASE

—Aromatic: 2-oxoglutarate aminotransferase has been purified about 950-fold from rat brain mitochondria. The purified enzyme was homogeneous in polyacrylamide gel electrophoresis and had a molecular weight of approx 63,000. On the basis of substrate specificity, substrate inhibition, purification ratio, yield, polyacrylamide gel electrophoresis and some other properties of the enzyme it has been suggested that brain mitochondrial tyrosine:2-oxoglutarate aminotransferase (l -tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) is identical with brain mitochondrial phenylalanine and kynurenine: 2-oxoglutarate aminotransferases (l -kynurenine: 2-oxoglutarate aminotransferase, EC 2.6.1.7), and also with aspartate: 2-oxoglutarate aminotransferase (l -aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1).     

Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH₃SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH₃SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH₃SeH and/or seleno-keto acid metabolites.     

Gene-enzyme relationships of aromatic acid biosynthesis in Bacillus subtilis

Mutants have been isolated which correspond to every step concerned with the biosynthesis of the aromatic amino acids in Bacillus subtilis. Each mutant has been characterized, and the lesion it bore was analyzed by deoxyribonucleic acid transformation and PBS-1 mediated transduction. The biochemical analysis revealed that each of the mutations appears to have affected a single enzyme, except for two groups of pleiotropic mutations. All aroF mutants (chorismic acid synthetase) lack dehydroquinic acid synthetase (aroB) activity. The gene that specifies aroB is closely linked to the gene coding for the aroF enzyme. Both genes are a part of the aro cluster. Mutants lacking chorismate mutase activity also lack d-arabino-heptulosonic acid-7-phosphate synthetase and shikimate kinase activity, presumably as a result of these three activities forming a multi-enzyme complex. Another mutant, previously undescribed, had been isolated. The affected gene codes for the tyrosine and phenylalanine aminotransferase activity. All of the mutations have been located on the B. subtilis genome except those in the genes specifying shikimate kinase activity and tyrosine-phenylalanine aminotransferase activity.     

Characterization of two multispecific aspartate aminotransferase isozymes purified from bushbean shoots capable of transaminating aromatic amino acids and severalDL-chloro-phenylalanines

Two electrophoretically distinct isozymes ofL-phenylalanine aminotransferase (Enz I, Enz II) purified from a total soluble shoot extract of bushbean have been characterized. The M_rs of Enz I and Enz II were 100 000 and 110 000, respectively. Both isozymes showed pH optima of 8.5. Enz I was able to use either 2-oxoglutarate (2-OG) or oxaloacetate (OAA) equally as a keto acid substrate whenL-phenylalanine was the amino donor, while Enz II preferred 2-OG. Neither isozyme was able to use glyoxylate or pyruvate in the presence ofL-phenylalanine. When tested with a range of protein amino acids, both Enz I and Enz II showed the highest rate of transamination withL-aspartate, indicating that both isozymes wereL-aspartate aminotransferases capable of also showingL-aromatic aminotransferase activity.L-Phenylalanine aminotransferase activity relative toL-aspartate aminotransferase activity was found to be 0.6 % for Enz I and 3.3% for Enz II. Lineweaver-Burk plots of kinetic data gave apparent K_m values (mM) for Enz I of 2.3 (L-Asp), 55.0 (L-Phe) and 9.0 (2-OG) and for Enz II, 2.8 (L-Asp), 320.0 (L-Phe) and 8.2 (2-OG). The values were confirmed by treatment of the data by Hill plots. When tested with a series of 12 ring-substitutedDL-chlorophenylalanines, Enz I was active only with the 3-chloro- and 4-chloro-compounds, while Enz II was active with all three monochloro-compounds as well as with the 2,4-, 2,6- and 3,4-dichlorophenylalanines. The activity of Enz II with 4-chlorophenylalanine was very high, 222 % higher than that observed withDL-phenylalanine. Enz I was completely inhibited by 1.0 mM Ca²⁺ while Enz II was unaffected by this cation, which suggested different subcellular locations for each isozyme. Cell fractionation studies indicated, however, that both Enz I and Enz II were cytoplasmic. Different isozymes of this multispecific aspartate—aromatic aminotransferase were found in the chloroplasts and mitochondria of bushbean shoots.