A brief history of the DNA repair field

The history of the repair of damaged DNA can be traced to the mid-1930s. Since then multiple DNA repair mechanisms, as well as other biological responses to DNA damage, have been discovered and their regulation has been studied. This article briefly recounts the early history of this field.     

The narrow substrate specificity of human tyrosine aminotransferase--the enzyme deficient in tyrosinemia type II

Human tyrosine aminotransferase (hTATase) is the pyridoxal phosphate-dependent enzyme that catalyzes the reversible transamination of tyrosine to p-hydrophenylpyruvate, an important step in tyrosine metabolism. hTATase deficiency is implicated in the rare metabolic disorder, tyrosinemia type II. This enzyme is a member of the poorly characterized Igamma subfamily of the family I aminotransferases. The full length and truncated forms of recombinant hTATase were expressed in Escherichia coli, and purified to homogeneity. The pH-dependent titration of wild-type reveals a spectrum characteristic of family I aminotransferases with an aldimine pK(a) of 7.22. I249A mutant hTATase exhibits an unusual spectrum with a similar aldimine pK(a) (6.85). hTATase has very narrow substrate specificity with the highest enzymatic activity for the Tyr/alpha-ketoglutarate substrate pair, which gives a steady state k(cat) value of 83 s(-1). In contrast there is no detectable transamination of aspartate or other cosubstrates. The present findings show that hTATase is the only known aminotransferase that discriminates significantly between Tyr and Phe: the k(cat)/K(m) value for Tyr is about four orders of magnitude greater than that for Phe. A comparison of substrate specificities of representative Ialpha and Igamma aminotransferases is described along with the physiological significance of the discrimination between Tyr and Phe by hTATase as applied to the understanding of the molecular basis of phenylketonuria.     

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.     

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.     

Kinetics of the alanine aminotransferase reaction in the mitochondrial and cell sap fractions of rat brain.

1. A reversible transamination reaction between L-glutamate and pyruvate, or L-alanine and 2-oxoglutarate, takes place in the mitochondrial and cell sap fractions of rat brain. 2. The maximum rate of the transamination reaction in both subfractions was observed in the presence of a keto- substrate concentration of 2.5 mM only, but an amino- donor concentration of 20 mM. 3. The apparent Menten-Michaelis constants for pyruvate and 2-oxoglutarate were of a 10(-4) M and for L-glutamate and L-alanine of a 10(-3) M order and were approximately the same for both fractions. 4. The ratio of the initial rate of the L-alanine + 2-oxoglutarate to the L-glutamate + pyruvate transamination reaction in the cell sap and mitochondrial fractions amounted to up to 2. 5. The apparent equilibrium constant derived from the Haldane equation was 7.01 for cell sap alanine aminotransferase and 4 for the mitochondrial enzyme. 6. Increasing pyridoxal-5'-phosphate concentrations in the incubation medium were accompanied by only non-significant stimulation of alanine aminotransferase activity in the mitochondrial and cell sap fractions. 7. A comparison of the kinetic data obtained on mitochondrial and cell sap alanine aminotransferases in vitro with the actual substrate concentrations in the transamination reaction in nervous tissue in vivo indicates that the direction of the transamination reaction in situ seems to be determined simply by compartmentation and by dynamic changes in amino- and keto- substrates in the mitochondrial and cell sap spaces.     

Pineapple Nematode Research in Hawaii: Past,Present, and Future

The first written record of pineapple in Hawaii is from 1813. In 1901 commercial pineapple production started, and in 1924 the Experiment Station for pineapple research was established. Nematode-related problems were recognized in the early 1900s by N. A. Cobb. From 1920 to approximately 1945 nematode management in Hawaiian pineapple was based on fallowing and crop rotation. During the 1920s and 1930s G. H. Godfrey conducted research on pineapple nematode management. In the 1930s and 1940s M. B. Linford researched biological control and described several new species of nematodes including Rotylenchulus reniformis. In 1941 nematology and nematode management were advanced by Walter Carter''s discovery of the first economical soil fumigant for nematodes, D-D mixture. Subsequently, DBCP was discovered and developed at the Pineapple Research Institute (PRI). Since 1945 soil fumigation has been the main nematode management strategy in Hawaiian pineapple production. Recent research has focused on the development of the nonvolatile nematicides, their potential as systemic nematicides, and their application via drip irrigation. Current and future research addresses biological and cultural alternatives to nematicide-based nematode management.     

Enzymatic synthesis of aminocyclitol moieties of aminoglycoside antibiotics from inositol by Streptomyces spp.: detection of glutamine-aminocyclitol aminotransferase and diaminocyclitol aminotransferase activities in a spectinomycin producer.

Extracts of stationary-phase mycelia of the spectinomycin producer Streptomyces flavopersicus ATCC 19756 catalyzed inositol dehydrogenase, L-glutamine:inosose aminotransferase, 2-epi-streptamine:inosose aminotransferase, streptamine:inosose aminotransferase, N3-methyl-2-deoxystreptamine:inosose aminotransferase, and aminodeoxy-scyllo-inositol:inosose aminotransferase reactions, as detected with a new rapid assay procedure. These results suggest that one or both amino groups of the N1,N3-dimethyl-2-epi-streptamine moiety of spectinomycin are derived by transamination from the alpha-amino group of L-glutamine. An enzymatic procedure for distinguishing among N1- and N3-monomethyl diaminocyclitol derivatives and their diaminocyclitol biosynthetic precursors is described. A scheme showing key roles of glutamine-aminocyclitol aminotransferases in biosynthesis of major aminoglycoside antibiotics is presented.     

A multispecific quintet of aromatic aminotransferases that overlap different biochemical pathways in Pseudomonas aeruginosa

Pseudomonas aeruginosa possesses dual enzymatic sequences to both L-phenylalanine and L-tyrosine, a biosynthetic arrangement further complicated by the presence of five aromatic aminotransferases. Each aminotransferase is capable of transamination in vitro with any of the three keto acid intermediates in the aromatic pathway (phenylpyruvate, 4-hydroxyphenylpyruvate, or prephenate). The fractional contribution of these aminotransferases to particular transamination reactions in vivo can best be approached through the systematic and sequential elimination of individual aminotransferase activities by mutation. A program of sequential mutagenesis has produced two aminotransferase-deficient mutations. The first mutation imposed a phenotype of bradytrophy for L-phenylalanine (doubling time of 2.4 h in minimal salts/glucose medium compared to a 1.0-h doubling time for wild type). This mutant completely lacked an enzyme denoted aminotransferase AT-2. A genetic background of aminotransferase AT-2 deficiency was used to select for a second mutation which produced a phenotype of multiple auxotrophy for L-phenylalanine, L-aspartate, and L-glutamate. The double mutant completely lacked activity for aromatic aminotransferase AT-1 in addition to the missing aminotransferase AT-2. Enzymes AT-1 (Mr = 64,000) and AT-2 (Mr = 50,000) were readily separated from one another by gel filtration and were individually characterized for pH optima, freeze-thaw stability, heat lability, and molecular weight. The phenotypic and enzymological characterizations of the aminotransferase mutants strongly support the primary in vivo role of enzyme AT-2 in L-phenylalanine and L-tyrosine biosynthesis, while enzyme AT-1 must primarily be engaged in L-aspartate and L-glutamate synthesis. The substrate specificities and possible in vivo functions for AT-3, AT-4, and AT-5 are also considered.     

A novel transaminase, (<Emphasis Type="Italic">R</Emphasis>)-amine:pyruvate aminotransferase,from <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. KNK168 (FERM BP-5228): purification,characterization, and gene cloning

A novel (R)-amine transaminase, which catalyzed (R)-enantioselective transamination of chiral amine, was purified to homogeneity from Arthrobacter sp. KNK168 (FERM BP-5228). The molecular mass of the enzyme was estimated to be 148 kDa by gel filtration and 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting a homotetrameric structure. The enzyme catalyzed transamination between amines and pyruvate stereo-specifically. The reaction on 1-methylbenzylamine was (R)-enantioselective. Pyruvate was the best amino acceptor, but the enzyme showed broad amino acceptor specificity for various ketone and aldehyde compounds. The apparent K _ms for (R)-1-methylbenzylamine and pyruvate were 2.62 and 2.29 mM, respectively. The cloned gene of the enzyme consists of an open reading frame (ORF) of 993 bp encoding a protein of 330 amino acids, with a calculated molecular weight of 36,288. The deduced amino acid sequence was found to be homologous to those of the aminotransferases belonging to fold class IV of pyridoxal-5′-phosphate-dependent enzymes, such as branched-chain amino acid aminotransferases.     

The biological standard of living in Indonesia during the 20th century: Evidence from the age at menarche

This article analyses long-term changes in the mean age at menarche (MAM) as a biological indicator of changes in the standard of living in Indonesia. It finds that MAM was about 15.5 for birth cohorts in the late-19th century, decreasing to 14.5 by the 1930s, at which level it stagnated until the gradual decrease resumed since the early 1960s to around 12.5 in the mid-2000s. The article considers that long-term improvements in nutrition, educational attainment and health care explain these trends. An international comparison of long-term changes finds that MAM in Indonesia was much lower than in Korea and China until respectively 1970 and 1990, but comparable to Japan until 1950 and to Malaysia until 1930. The article presents reasons why these differences are unlikely to be related to dissimilarities in climate and ethnicity, and concludes that they are indicative of relative standards of living.     

Alanine aminotransferase and glycine aminotransferase from maize (Zea mays L.) leaves.

Alanine aminotransferase (AlaAT, EC 2.6.1.2) and glycine aminotransferase (GlyAT, EC 2.6.1.4), two different enzymes catalyzing transamination reactions with L-alanine as the amino-acid substrate, were examined in maize in which alanine participates substantially in nitrogen transport. Preparative PAGE of a partially purified preparation of aminotransferases from maize leaves gave 6 fractions differing in electrophoretic mobility. The fastest migrating fraction I represents AlaAT specific for L-alanine as amino donor and 2-oxoglutarate as amino acceptor. The remaining fractions showed three aminotransferase activities: L-alanine-2-oxoglutarate, L-alanine-glyoxylate and L-glutamate-glyoxylate. By means of molecular sieving on Zorbax SE-250 two groups of enzymes were distinguished in the PAGE fractions: of about 100 kDa and 50 kDa. Molecular mass of 104 kDa was ascribed to AlaAT in fraction I, while the molecular mass of the three enzymatic activities in 3 fractions of the low electrophoretic mobility was about 50 kDa. The response of these fractions to: aminooxyacetate, 3-chloro-L-alanine and competing amino acids prompted us to suggest that five out of the six preparative PAGE fractions represented GlyAT isoforms, differing from each other by the L-glutamate-glyoxylate:L-alanine-glyoxylate:L-alanine-2-oxoglutarate activity ratio.     

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.     

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.     

An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavor compounds.

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be involved in the complex process of cheese flavor development. In lactococci, transamination is the first step in the degradation of aromatic and branched-chain amino acids which are precursors of aroma compounds. Here, the major aromatic amino acid aminotransferase of a Lactococcus lactis subsp. cremoris strain was purified and characterized. The enzyme transaminates the aromatic amino acids, leucine, and methionine. It uses the ketoacids corresponding to these amino acids and alpha-ketoglutarate as amino group acceptors. In contrast to most bacterial aromatic aminotransferases, it does not act on aspartate and does not use oxaloacetate as second substrate. It is essential for the transformation of aromatic amino acids to flavor compounds. It is a pyridoxal 5'-phosphate-dependent enzyme and is composed of two identical subunits of 43.5 kDa. The activity of the enzyme is optimal between pH 6.5 and 8 and between 35 and 45 degrees C, but it is still active under cheese-ripening conditions.     

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.     

Crystal structures of aminotransferases Aro8 and Aro9 from Candida albicans and structural insights into their properties

Aminotransferases catalyze reversibly the transamination reaction by a ping-pong bi-bi mechanism with pyridoxal 5′-phosphate (PLP) as a cofactor. Various aminotransferases acting on a range of substrates have been reported. Aromatic transaminases are able to catalyze the transamination reaction with both aromatic and acidic substrates. Two aminotransferases from C. albicans, Aro8p and Aro9p, have been identified recently, exhibiting different catalytic properties. To elucidate the multiple substrate recognition of the two enzymes we determined the crystal structures of an unliganded CaAro8p, a complex of CaAro8p with the PLP cofactor bound to a substrate, forming an external aldimine, CaAro9p with PLP in the form of internal aldimine, and CaAro9p with a mixture of ligands that have been interpreted as results of the enzymatic reaction. The crystal structures of both enzymes contains in the asymmetric unit a biologically relevant dimer of 55?kDa for CaAro8 and 59?kDa for CaAro9p protein subunits. The ability of the enzymes to process multiple substrates could be related to a feature of their architecture in which the active site resides on one subunit while the substrate-binding site is formed by a long loop extending from the other subunit of the dimeric molecule. The separation of the two functions to different chemical entities could facilitate the evolution of the substrate-binding part and allow it to be flexible without destabilizing the conservative catalytic mechanism.     

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

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

Effect of trophic conditions and humic acid on alanine transamination in wheat plants

In dialyzed extracts from winter wheat plants an intensive enzymatical transamination reaction occurred between L-α-alanine and α-ketoglutaric acid (L-α-alanine + + 2-oxoglutarate = pyruvate + L-glutamate, EC 2.6.1.2) as well as a weak nonenzymatical transamination reaction, practically immeasurable. Pyridoxal-5’-phosphate strongly affected the reaction rate. Besides the transamination product-glutamate, γ-aminobutyric acid was formed in this reaction. This amino acid could have originated neither via proteolysis of the enzyme extract, nor via decarboxylation from glutamate formed, nor via transamination of succinic-γ-semialdehyde after α-ketoglutarate decarboxylation. This was the only case of its formation in the transamination reactions investigated in our laboratory — it originated from the alanine-glutamate reaction only, and the mechanism of its biosynthesis cannot be elucidated for the present. Dialysates from shoots exhibited a significantly higher enzymatic activity in comparison with those from roots. The effect of trophic conditions (Knop’s nutrient solution, a water solution of potassium humate, water) was not revealed when calculating per dry weight unit. However, when calculating per protein unit an increased activity was found in the dialysates from shoots of both nutrient — deficient variants. Roots of plants cultivated in potassium humate had the lowest activity. The discussion concerns the possibility of an adaptive use of this transamination for increasing the essential glutamate level in green parts of the plants cultivated under unfavourable nutritive conditions, and also deals with a further characteristic of the differing metabolism of plants cultivated in humate.     

Molecular cloning, expression and characterization of pyridoxamine-pyruvate aminotransferase

Pyridoxamine-pyruvate aminotransferase is a PLP (pyridoxal 5'-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine-pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the alpha family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429-432]. The K(d) value for pyridoxal determined by means of CD was 100-fold lower than the K(m) value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed.     

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.