AROMATIC AMINOTRANSFERASE ACTIVITY OF RAT BRAIN CYTOPLASMIC AND MITOCHONDRIAL ASPARTATE AMINOTRANSFERASES

Abstract— Mitochondrial and cytoplasmic forms of aspartate aminotransferase were purified from rat brain homogenates and tested for their ability to catalyze transamination of various aromatic amino acids. The mitochondrial enzyme exhibited activity toward tyrosine and phenylalanine with 2-oxoglutar-ate as acceptor, although the specific activities were less than 1% of the corresponding aspartate activity when all substrates were 10 mM. Even less activity was seen with DOPA, 5-hydroxytryptophan and tryptophan. The cytoplasmic aspartate aminotransferase was active toward tryptophan, 5-hydroxytryptophan and DOPA, but these transaminations were favored by pyruvate or oxaloacetate rather than 2-oxoglutarate as keto acid. Based on co-migration of aromatic activities with the respective aspartate aminotransferases during isoelectric focusing and based on equal sensitivities of aromatic transamination and aspartate transamination to inhibition by vinylglycine, it was concluded that all activities resided in the aspartate aminotransferase enzymes. Some doubt exists, however, as to the physiological significance of these alternate activities in view of the requirement that aromatic amino acids must compete with aspartate for transamination by these enzymes.     

Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis.

Although several high-resolution X-ray crystallographic structures have been determined for Escherichia coli aspartate aminotransferase (eAATase), efforts to crystallize E. coli tyrosine aminotransferase (eTATase) have been unsuccessful. Sequence alignment analyses of eTATase and eAATase show 43% sequence identity and 72% sequence similarity, allowing for conservative substitutions. The high similarity of the two sequences indicates that both enzymes must have similar secondary and tertiary structures. Six active site residues of eAATase were targeted by homology modeling as being important for aromatic amino acid reactivity with eTATase. Two of these positions (Thr 109 and Asn 297) are invariant in all known aspartate aminotransferase enzymes, but differ in eTATase (Ser 109 and Ser 297). The other four positions (Val 39, Lys 41, Thr 47, and Asn 69) line the active site pocket of eAATase and are replaced by amino acids with more hydrophobic side chains in eTATase (Leu 39, Tyr 41, Ile 47, and Leu 69). These six positions in eAATase were mutated by site-directed mutagenesis to the corresponding amino acids found in eTATase in an attempt to redesign the substrate specificity of eAATase to that of eTATase. Five combinations of the individual mutations were obtained from mutagenesis reactions. The redesigned eAATase mutant containing all six mutations (Hex) displays second-order rate constants for the transamination of aspartate and phenylalanine that are within an order of magnitude of those observed for eTATase. Thus, the reactivity of eAATase with phenylalanine was increased by over three orders of magnitude without sacrificing the high transamination activity with aspartate observed for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Kinetic studies with the use of proton-magnetic-resonance spectroscopy of the specific alpha-deuteration of amino acids by Escherichia coli aspartate aminotransferase.

Escherichia coli aspartate aminotransferase was exposed to aspartate or phenylalanine without oxo acid in buffered 2H2O. The alpha-hydrogen of the amino acids underwent first-order exchange with respect to both substrate and enzyme. P.m.r. spectroscopy gave consistent reaction-rate constants. The deuterium-exchange rate was only moderately increased by addition of oxo acids and was of the same order as the transamination rate. No beta-deuteration was observed. The C(alpha)-H-bond-breaking step is discussed as a part of the entire transamination mechanism.     

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.     

An in vivo function of glucagon-lnduced phenylalanine: Pyruvate transaminase in p-chlorophenylalanine-treated rats

The effect of glucagon-induced phenylalanine:pyruvate transaminase on the urinary excretion of the unconjugated metabolites of phenylalanine transamination was studied in rats. Chronic injection of glucagon induced an 18-fold increase in hepatic phenylalanine:pyruvate transaminase activity. Treatment with p-chlorophenylalanine (PCPA) blocked phenylalanine hydroxylase and caused an elevation of plasma phenylalanine following administration of an intraperitoneal loading dose of this amino acid. Gasliquid Chromatographic analysis demonstrated the presence of phenylpyruvate, phenyllactate, and O-hydroxyphenylacetate in the urine of PCPA- and PCPA-glucagontreated rats, but not control or glucagon-treated animals. Combined PCPA-glucagon treatment caused twofold increase in phenylpyruvate and phenyllactate concentrations and a fivefold increase in O-hydroxyphenylacetate concentration, when compared to urinary metabolite levels from rats receiving only PCPA treatment. A decrease in plasma phenylalanine was found together with the elevated urinary levels of the phenylalanine transamination metabolites. The results provide the first evidence that the unconjugated transamination metabolite concentrations increase when concurrent treatment with glucagon causes high-level induction of hepatic phenylalanine:pyruvate transaminase.     

Rerouting Citrate Metabolism in Lactococcus lactis to Citrate-Driven Transamination

Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049-4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases of L. lactis accept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transamination in vivo was further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate.     

Glutamine transaminase from brain tissue. Further studies on kinetic properties and specificity of the enzyme.

Glutamine transaminase, highly purified from rat brain, was studied. In the first series of experiments, the kinetics of the transamination reaction between 2-oxoglutaramate and phenylalanine were examined in order to determine the type of reaction mechanism. This proved to be of the ping-pont type, as can be expected for a transamination. The specificity of the enzyme for various amino acids and 2-oxo acids was then studied in detail. The most active substrates were glutamine, methionine and ethionine as amino-group donors, and phenylpyruvate, glyoxalate and 2-oxo-4-methiobutyrate as amino-group acceptors. For these and several other substrates, the kinetic constants, V and Km, were determined.     

The use of natural and unnatural amino acid substrates to define the substrate specificity differences of Escherichia coli aspartate and tyrosine aminotransferases.

The tyrosine (eTATase) and aspartate (eAATase) aminotransferases of Escherichia coli transaminate diacarboxylic amino acids with similar rate constants. However, eTATase exhibits approximately 10(2)-10(4)-fold higher second-order rate constants for the transamination of aromatic amino acids than does eAATase. A series of natural and unnatural amino acid substrates was used to quantitate specificity differences for these two highly related enzymes. A general trend toward lower transamination activity with increasing side-chain length (extending from aspartate to glutamate to alpha-aminoadipate) is observed for both enzymes. This result suggests that dicarboxylate ligands associate with the two highly related enzymes in a similar manner. The high reactivity of the enzymes with L-Asp and L-Glu can be attributed to an ion pair interaction between the side-chain carboxylate of the amino acid substrate and the guanidino group of the active site residue Arg 292 that is common to both enzymes. A strong linear correlation between side-chain hydrophobicity and transamination rate constants obtains for n-alkyl side-chain amino substrates with eTATase, but not for eAATase. The present kinetic data support a model in which eAATase contains one binding mode for all classes of substrate, whereas the active site of eTATase allows an additional mode that has increased affinity for hydrophobic amino acid.     

Asparagine transaminase from rat liver.

Asparagine transaminase has been purified about 200-fold from rat liver. The enzyme has a broad specificity toward both amino acids and alpha-keto acids. Thus, amino acids substituted in the beta position such as asparagine, S-methylcysteine, phenylalanine, cysteine, serine, and aspartate are substrates. The enzyme is also active with alanine, methionine, homoserine, alpha-aminobutyrate, glutamine, and leucine. The enzyme has a high affinity for glyoxylate but the affinity falls off markedly through the series glyoxylate, pyruvate, alpha-ketoburyrate, alpha-Keto acids substituted in the beta or gamma position, such as alpha-ketosuccinamate, phenylpyruvate, p-hydroxyphenylpyruvate, alpha-keto-gamma-methiolburyrate, and alpha-keto-gamma-hydroxybutyrate, are substrates for the enzyme. Amino acids or alpha-keto acids possessing a branch point at the beta carbon are inactive. Kinetic analysis of the asparagine glyoxylate transamination reaction is consistent with a ping-pong mechanism.     

Aminotransferases in the bivalve mollusc Mytilus edulis L. and short term effects of crude oil in brackish water

Transaminases are among the crucial enzymes in amino acid metabolism, which in aquatic organisms is known to be affected by exposure to oil hydrocarbons. The transamination reactions in Mytilus edulis L. were studied to estimate their adequacy to indicate short term oil exposure in mussels. The transamination reactions were measured using paper chromatography and spectrophotometry. A high degree of transamination was observed between 2 oxoglutarate and alanine, aspartate and ornithine. A slight degree of transamination was shown with methionine, leucine, isoleucine, phenylalanine, serine, tryptophan, threonine, tyrosine and valine. No transamination was observed between 2 oxoglutarate and glycine, arginine, histidine, lysine, proline, citrulline and alanine. The effect of the water accommodated fraction WAF of crude oil on selected transaminase reactions was measured. The highest changes during the WAF exposure were mostly observed in the gills and mantle. Alanine aminotransferase EC 2.6.1.2 activity in the mantle was, at its highest, 55 over the control. Aspartate aminotransferase EC 2.6.1.1 activity increased in the gills by 52 . For ornithine transamination, in the gills the highest increase was by 75 and in the mantle by 50 . The metabolic pathways involved in the alterations of aminotransferase activities are discussed. It is concluded that ornithine transamination in gills is a potential indicator for short term crude oil exposure in Mytilus edulis. More studies are needed to evaluate the effects of other organic pollutants on ornithine transamination.     

Aminotransferases in the bivalve mollusc Mytilus edulis L. and short term effects of crude oil in brackish water

Transaminases are among the crucial enzymes in amino acid metabolism, which in aquatic organisms is known to be affected by exposure to oil hydrocarbons. The transamination reactions in Mytilus edulis L. were studied to estimate their adequacy to indicate short term oil exposure in mussels. The transamination reactions were measured using paper chromatography and spectrophotometry. A high degree of transamination was observed between 2 oxoglutarate and alanine, aspartate and ornithine. A slight degree of transamination was shown with methionine, leucine, isoleucine, phenylalanine, serine, tryptophan, threonine, tyrosine and valine. No transamination was observed between 2 oxoglutarate and glycine, arginine, histidine, lysine, proline, citrulline and alanine. The effect of the water accommodated fraction WAF of crude oil on selected transaminase reactions was measured. The highest changes during the WAF exposure were mostly observed in the gills and mantle. Alanine aminotransferase EC 2.6.1.2 activity in the mantle was, at its highest, 55 over the control. Aspartate aminotransferase EC 2.6.1.1 activity increased in the gills by 52 . For ornithine transamination, in the gills the highest increase was by 75 and in the mantle by 50 . The metabolic pathways involved in the alterations of aminotransferase activities are discussed. It is concluded that ornithine transamination in gills is a potential indicator for short term crude oil exposure in Mytilus edulis. More studies are needed to evaluate the effects of other organic pollutants on ornithine transamination.     

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.     

Enzyme-induced inactivation of transminases by acetylenic and vinyl analogues of 4-aminobutyrate.

The reactions of two analogues of 4-aminobutyrate, namely 4-aminohex-5-ynoate and 4-aminohex-5-enoate, with three transaminases were studied. Three pure enzymes were used, aminobutyrate transaminase (EC 2.6.1.19), ornithine transaminase (EC 2.6.1.13) and aspartate transaminase (EC 2.6.1.1), and the course of the reactions was studied by observing changes in the absorption spectrum of the bound coenzyme and by observing loss of activity. All of the enzymes were inactivated by either inhibitor, but amino-hexenoate showed a marked specificity for aminobutyrate transaminase. Aminohexynoate was most potent towards ornithine transaminase, and with this enzyme transamination of the inhibitor is an important factor in protecting the enzyme. Most of the reactions could be analysed as first order, with the observed rate constant showing a hyperbolic dependence on inhibitor concentration.     

Mechanism of racemization of amino acids by aspartate aminotransferase.

Aspartate aminotransferase (mitochondrial isoenzyme from chicken) has been found to racemize very slowly dicarboxylic amino acid substrates in the presence of their cognate oxo acids [Kochhar, S. & Christen, P. (1988) Eur. J. Biochem. 175, 433-438]. Tyrosine, phenylalanine and alanine are racemized at the same rate although they undergo the transamination reaction 3-5 orders of magnitude more slowly than the dicarboxylic substrates. Similarly, the truncated enzyme aspartate aminotransferase-(27/32-410) catalyzes the racemization at the same rate as the native enzyme, while its rate of transamination is decreased to 3% of that of the native enzyme. Apparently, the rate-limiting step in racemization is not immediately linked to the transamination cycle. Decreasing the water concentration in the reaction medium by adding methanol at 0 degrees C drastically reduces the rate of racemization without affecting the rate of transamination. On the basis of these and additional kinetic data and the model of the three-dimensional structure of the active site, we conclude that a water molecule is responsible for the protonation of C alpha of the coenzyme-substrate intermediate from the wrong side. The diffusion of the water molecule into the interior of the enzyme appears to be the rate-limiting step in aspartate-aminotransferase-catalyzed racemization.     

Novel biosynthetic routes to non-proteinogenic amino acids as chiral pharmaceutical intermediates

Transaminases catalyse the reversible transfer of amino and keto groups between an amino acid and keto acid substrate pair. Many bacterial transaminases accept a wide array of keto acids as amino acceptors and are useful as commercial biocatalysts in the preparation of amino acids. Since the reaction equilibrium typically lies close to unity, several approaches have been described to improve upon the 50% product yield, using additional enzymes. The present work describes an efficient means to significantly increase product yield in transamination using the aromatic transaminase of Escherichia coli encoded by the tyrB gene, with -aspartate as the amino donor. This is achieved by the introduction of the alsS gene encoding the acetolactate synthase of Bacillus subtilis, which eliminates pyruvate and alanine produced as a by-product of aspartate transamination. The biosynthesis of the non-proteinogenic amino acid -2-aminobutyrate is described using a recombinant strain of E. coli containing the cloned tyrB and alsS genes. The strain additionally carries the cloned ilvA gene of E. coli encoding threonine deaminase to produce the substrate 2-ketobutyrate from -threonine. An alternate coupled process uses lysine -aminotransferase in concert with a transaminase using -glutamate as the amino donor.     

Rapid Inactivation of Brain Glutamate Decarboxylase by Aspartate

In the absence of its cofactor, pyridoxal 5'-phosphate (pyridoxal-P), glutamate decarboxylase is rapidly inactivated by aspartate. Inactivation is a first-order process and the apparent rate constant is a simple saturation function of the concentration of aspartate. For the beta-form of the enzyme, the concentration of aspartate giving the half-maximal rate of inactivation is 6.1 +/- 1.3 mM and the maximal apparent rate constant is 1.02 +/- 0.09 min-1, which corresponds to a half-time of inactivation of 41 s. The rate of inactivation by aspartate is about 25 times faster than inactivation by glutamate or gamma-aminobutyric acid (GABA). Inactivation is accompanied by a rapid conversion of holoenzyme to apoenzyme and is opposed by pyridoxal-P, suggesting that inactivation results from an alternative transamination of aspartate catalyzed by the enzyme, as previously observed with glutamate and GABA. Consistent with this mechanism pyridoxamine 5'-phosphate, an expected transamination product, was formed when the enzyme was incubated with aspartate and pyridoxal-P. The rate of transamination relative to the rate of decarboxylation was much greater for aspartate than for glutamate. Apoenzyme formed by transamination of aspartate was reactivated with pyridoxal-P. In view of the high rate of inactivation, aspartate may affect the level of apoenzyme in brain.     

Analysis of conformationally restricted alpha-ketoglutarate analogues as substrates of dehydrogenases and aminotransferases.

Five synthetic, conformationally restricted alpha-ketoglutarate analogues were tested as substrates of a variety of dehydrogenases and aminotransferases. The compounds were found not to be detectable substrates of glutamate dehydrogenase, L-leucine dehydrogenase, L-phenylalanine dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glutamine transaminase K, aspartate aminotransferase, alanine aminotransferase, and alpha-ketoglutarate dehydrogenase complex. However, two thermostable aminotransferases were identified that catalyze transamination between several L-amino acids (e.g., phenylalanine, glutamate) and the alpha-ketoglutarate analogues of interest. Transamination between L-glutamate (or L-phenylalanine) and the alpha-ketoglutarate analogues was found to be 0.13 to 1.08 micromol/h/mg at 45 degrees C. The products resulting from transamination between L-phenylalanine and the alpha-ketoglutarate analogues were separated by reverse-phase HPLC, and the newly formed amino acid analogues were analyzed by LC-MS in an ion selective mode. In each case, the ions obtained were consistent with the expected product and a representative example is provided. The possibility existed that although the alpha-ketoglutarate analogues are not substrates of the dehydrogenases and most of the aminotransferases investigated, they might be good inhibitors. Weak inhibition of aminotransferases and glutamate dehydrogenase was found with some of the alpha-ketoglutarate analogues. The newly available thermostable aminotransferases may have general utility in the synthesis of bulky L-amino acids from the corresponding alpha-keto acids.     

TRANSAMINATIONS BETWEEN AMINO ACIDS AND KETO ACIDS ELEVATED IN PHENYLKETONURIA AND MAPLE SYRUP URINE DISEASE

Abstract— The transamination between amino acids and aliphatic and aromatic keto acids has been investigated in homogenates of human and rat brain. Tryptophan, phenylalanine and 3,4-dihydroxyphenylalanine (DOPA) at concentrations of 3.6 min and below trans-aminated aromatic keto acids more rapidly than α-ketoglutarate; lower K_m values were found for tryptophan and phenylalanine in the presence of the aromatic keto acid. Rat brain and liver arninotransferases exhibited similar affinities for tryptophan in the presence of different keto acids. Branched chain keto acids were also acceptors of the amino groups of tryptophan and DOPA. In brain homogenates α-ketoglutarate and p -hydroxyphenyl-pyruvate were transaminated by tyrosine and 5-hydroxytryptophan at about equal rates, whereas a-ketoglutarate was transaminated more rapidly with aliphatic amino acids. At concentrations of 1.6 m DOPA and 0.8 mM p -hydroxyphenylpyruvate, transamination was 6-fold greater than the rate of formation of dopamine. The dihydroxyphenylpyruvate formed during arninotransfer from DOPA by brain tissue was not readily decarboxylated, whereas 65–70 per cent of the indolepyruvate formed from tryptophan was decarboxylated. We suggest that an increased rate or degree of transamination between tryptophan and aromatic and branched chain keto acids may explain the increased excretion of non-hydroxylated indolic acids in phenylketonuria and'maple syrup urine'disease, respectively. Increased aminotransfers from tryptophan and DOPA may reduce the amounts of precursors available for the synthesis of serotonin and catecholamines, both of which are at low levels in the sera of untreated phenylketonurics.     

Histidine:pyruvate transamination and phyenylalanine:pyruvate transamination may be catalyzed by the same enzyme.

Some properties of histidine:pyruvate transaminase (HPT) and phenylalanine:pyruvate transaminase (PPT) in the cytosol of rat liver were studied. HPT and PPT activity could not be separated by DEAE-Sephadex A-50 or hydroxylapatite column chromatography, and the ratio of $\text{HPT}\text{PPT}$ activity remained constant during these purification procedures. The two enzyme activities also showed similar heat stability and responses to glucagon injection. Based on these findings, we suggest that a single enzyme may specifically catalyze histidine:pyruvate and phenylalanine:pyruvate transamination.