Aspartic acid with transaminases

Aspartic acid, alanine, phenylalanine, and lysine were manufactured by enzymatic route. Immobilized E. coli cells expressing aspartate or the immobilized enzyme has been used in the commercial production of aspartic acid from ammonia and fumaric acid. Chibata and coworkers also produced alanine by microbial Pseudomonas dacunhae) L-aspartate P-decarboxylase with aspartate as the starting material. Phenyl alanine was manufactured from fw s-cinnamic acid and ammonia by the enzymatic route by phenyl alanine ammonia lyase as catalyst or from phenyl pyruvate and aspartic acid using transaminase. 

In all cases the keto acids seem to be formed by typical a-ketogluta-rate-linked, pyridoxal phosphate-dependent transaminases (EC 2.6.1.6, etc.) (9, 154, 156, 157). There has been little study of isolated, presumably specific enzymes in connection with flavors, although the leucine and alanine aminotransferases of tomato have been precipitated with (NH4)oS04 (164, 165). Transaminase activity in Saccharomyces cere-visiae has a pH optimum of 7.2 (154), and a-ketoglutarate is the only amino group recipient (154, 166). Only aspartate and amino acids with hydrophobic side chains are acted on (154). 

A number of amino acids, like alanine, leucine, tyrosine, aspartic acid, cystein and arginine react with a-ketoacids and transfer their a-amino group to the a-carb-on of the a-keto acids. These reactions are catalysed by the enzyme called transaminase or aminotransferase. For example, transfer of the amino group of aspartic acid (14) to a-ketoglutaric acid (23) gives glutamic acid (16) and oxaloacetic acid (24). 

The synthesis of chiral a-amino acids starting from a-keto acids by means of a transamination has been reported by NSC Technologies [26, 27]. In this process, which can be used for the preparation of l- as well as D-amino acids, an amino group is transferred from an inexpensive amino donor, e.g., L-glutamic acid, l-22, or L-aspartic acid, in the presence of a transaminase (= aminotransferase). This reaction requires a cofactor, most commonly pyridoxal phosphate, which is bound to the transaminase. The substrate specificity is broad, allowing the conversion of numerous keto acid substrates under formation of the L-amino acid products with high enantioselectivities [28]. 

S )-Enantiomcrs of fluorophcnylalanincs and 4-(trifluoromethyl)phenylalanine were successfully prepared from the corresponding 2-oxo acids by the transfer of an amino group from (S)-aspartic acid catalyzed by a specific transaminase of microbial origin20. The biomimetic reduction of other imines with NAD coenzymes has also been described21 28. 

A further synthesis of labeled aspartic acids 2Ia used the enzyme aconitate isomerase (EC 5.3.3.7). This enzyme interconverts cis- and frans-aconitates 238 and 239, respectively, with exchange of the 4-pro-S hydrogen. The (4S)-[4- Hi]-trans-aconitate 239, H = H, produced in H20 could be ozonized and then converted in situ with sodium periodate and glutamic oxaloacetic transaminase (EC 2.6.1.2) to (3S)-[3- H,]aspartic acid 21a, (223). This process involves (3S)-[3- Hi]oxaloacetic acid 240, Ha = H, as an intermediate. 

Commercial preparations of pig heart glutamate-oxaloacetate transaminase have been screened for their ability to transaminate various a-keto acids with l-[ N]glutamate (32). In addition to l-[ N]aspartate, enzyme preparations were able to catalyze the formation of labeled tyrosine, phenylalanine, leucine, and dihydroxyphenylalanine, as demonstrated by HPLC (17). However, these amino acids have not yet been obtained in radiopure form by this method. The -keto acid analogs of valine and tryptophan were not transaminated by the enzyme preparations. Glutamate-oxaloacetate transaminases obtained from several commercial sources have varying abilities to transaminate the -keto acid... 

A possible explanation for the superiority of the amino donor, L-aspartic add, has come from studies carried out on mutants of E. coli, in which only one of the three transaminases that are found in E. coli are present. It is believed that a branched chain transaminase, an aromatic amino add transaminase and an aspartate phenylalanine aspartase can be present in E. coli. The reaction of each of these mutants with different amino donors gave results which indicated that branched chain transminase and aromatic amino add transminase containing mutants were not able to proceed to high levels of conversion of phenylpyruvic add to L-phenylalanine. However, aspartate phenylalanine transaminase containing mutants were able to yield 98% conversion on 100 mmol l 1 phenylpyruvic acid. The explanation for this is probably that both branched chain transaminase and aromatic amino acid transminase are feedback inhibited by L-phenylalanine, whereas aspartate phenylalanine transaminase is not inhibited by L-phenylalanine. In addition, since oxaloacetate, which is produced when aspartic add is used as the amino donor, is readily converted to pyruvic add, no feedback inhibition involving oxaloacetate occurs. The reason for low conversion yield of some E. coli strains might be that these E. cdi strains are defident in the aspartate phenylalanine transaminase. 

When administering the HMG-CoA reductase inhibitors and the fibric acid derivatives, the nurse monitors the patient s fiver function by obtaining serum transaminase levels before the drug regimen is started, at 6 and 12 weeks, then periodically thereafter because of the possibility of liver dysfunction with the drugs. If aspartate aminotransferase (AST) levels increase to three times normal, the primary care provider in notified immediately because the HMG-CoA reductase inhibitor therapy may be discontinued. 

This enzyme [EC 2.6.1.21], also known as D-aspartate aminotransferase, D-amino acid aminotransferase, and D-amino acid transaminase, catalyzes the reversible pyridoxal-phosphate-dependent reaction of D-alanine with a-ketoglutarate to yield pyruvate and D-glutamate. The enzyme will also utilize as substrates the D-stereoisomers of leucine, aspartate, glutamate, aminobutyrate, norva-hne, and asparagine. See o-Amino Acid Aminotransferase... 

Addition of ethyl acetate to a specimen having a transaminase activity of 47 units was responsible for the following increases in enzyme activity 10 mg/100 ml, 60 units 20 mg/100 ml, 77 units 40 mg/100 ml, 107 units and 80 mg/100 ml, 150 units. Transaminase activity in these specimens determined by another method ranged from 32 to 34 units (C7). Thus, when serum from patients with ketosis is assayed for aspartate aminotransferase activity by the diazo method, false elevations of activity may be recorded due to reaction of acetoacetic acid. In Table 11 are shown some values obtained by the diazo method and by an ultraviolet NADH NAD aspartate aminotransferase technique (B12). Examination of the medical records of these patients indicated that they were either diabetics who were in ketosis or individuals who were eating very poorly and had some degree of starvation ketosis. Similar elevations have been observed in patients receiving p-aminosalicylic acid (G6). 

A 61-year-old man with epilepsy had altered consciousness after his dose of valproate was increased because of poor seizure control. Electroencephalography showed triphasic waves and high-amplitude delta-wave activity with frontal predominance. Although serum aspartate transaminase and alanine transaminase were normal, the serum ammonium concentration was high at 960 ng/ml (reference range 30-470). Serum amino acid analysis showed multiple minor abnormalities. Valproate was withdrawn. He improved within 4 days... 

In the presence of NAD, L-malic acid is oxidised to oxaloacetate in a reaction catalysed by L-malate deshydrogenase (l-MDH). The reaction equilibrium is forced in the direction of the products by the elimination of oxaloacetate, via its reaction with 1-glutamate, resulting in the production of L-aspartate. This reaction is catalysed by glutamate-oxaloacetate-transaminase (GOT) ... 

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