Amino donor

One class of enzymes that follow a ping-pong-type mechanism are aminotransferases (previously known as transaminases). These enzymes catalyze the transfer of an amino group from an amino acid to an a-keto acid. The products are a new amino acid and the keto acid corresponding to the carbon skeleton of the amino donor ... 

FIGURE 14.22 Glutamate aspartate aminotransferase, an enzyme conforming to a double-displacement bisnbstrate mechanism. Glutamate aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme. The pyridoxal serves as the —NH, acceptor from glntamate to form pyridoxamine. Pyridoxamine is then the amino donor to oxaloacetate to form asparate and regenerate the pyridoxal coenzyme form. (The pyridoxamine enzyme is the E form.)... 

The elimination of the amino donor, L-aspartic acid, resulted in an almost complete reduction of activity. Neither cell permeabilisation nor cofactor (pyridoxalphosphate) addition were essential for L-phenylalanine production. Maximum conversion yield occurred (100%, 22 g r) when the amino donor concentration was increased. Aspartic add was a superior amino donor to glutamic add 35 g l 1 was used. 

The final conversion yield decreased when substrate concentration was increased from 2% to 4%. This was attributed to end product inhibition by the L-phenylalanine produced. Thus although faster conversion rates were observed with addition of high substrate concentrations, the product titres never exceeded 16 g l1. As already discussed the rate of yield of the conversion was proportional to the concentration of amino donor employed. Using a ratio of 1 3 substrate to amino donor, almost a 90% conversion was achieved in 3 hours. 

The results obtained with the P. fluorescerts strain without biochemical manipulation compare well with those reported for a E. cdi strain. Both achieve volumetric rates of 3 g T1 h 1 under normal conditions. So it appears that the efficiency of the process can be increased by a few simple operations increasing the pH and amino donor concentration (aspartic add). 

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. 

To establish the most advantageous conditions for production of L-phenylalanine from acetamidocinnamic add using two micro-organisms the following factors were investigated pH, amino donor and ratio of two enzyme activities. 

The best results were obtained with L-aspartic add as the amino donor for P. denitrificam and phenylpyruvic add as the amino acceptor. With L-aspartic add, conversion of phenylpyruvic add exceeded 90%. This may be attributed to absence of feedback inhibition of the reaction due to metabolism of file reaction product, oxaloacetic add. When using glutamic acid the conversion of phenylpyruvic add did not exceed 60%. 

A new development is the industrial production of L-phenylalanine by converting phenylpyruvic add with pyridoxalphosphate-dependent phenylalanine transaminase (see Figure A8.16). The biotransformation step is complicated by an unfavourable equilibrium and the need for an amino-donor (aspartic add). For a complete conversion of phenylpyruvic add, oxaloacetic add (deamination product of aspartic add) is decarboxylated enzymatically or chemically to pyruvic add. The use of immobilised . coli (covalent attachment and entrapment of whole cells with polyazetidine) is preferred in this process (Figure A8.17). 

Figure 28-3. Formation of alanine by transamination of pyruvate. The amino donor may be glutamate or aspartate. The other product thus is a-ketoglutarate or oxaloacetate.

Transaminase enzymes (also called aminotransferases) specifically use 2-oxoglutarate as the amino group acceptor to generate glutamate but some have a wide specificity with respect to the amino donor. For example, the three branched-chain amino acids leucine, isoleucine and valine, all serve as substrates for the same enzyme, branched-chain amino acid transaminase, BCAAT ... 

The same group have used the enzyme combination employed in the aspartate deracemization cited above to deracemize 2-naphthylalanine, hut have made use of an interesting innovation introduced by Helaine et al to pull over the poised equilibrium of the transamination reaction. Cysteine sulphinic acid was used as the amino donor in the transamination. The oxoacid product spontaneously decomposes in to pyruvic acid and SO2 (Scheme 3). 

The divergent pathways arise from selective enolization. Thus, in media of lower acidity enolization occurs toward C-3, whereas in a stronger acid complete N-pro-tonation shifts the enolization toward C-l. The amino donor confers certain acceptor character C-l (although this is a captodative situation), but upon protonation it is converted into an acceptor and its adjacent carbon atom, a donor. 

Acetamidodeoxyhexoses. A further modification of the 4-keto-inter-mediate has been independently shown by Ashwell and by Strominger and associates (Table I, References 20, 21, 22, 23). Transamination reactions with L-glutamate as the amino donor and pyridoxal phosphate as coenzyme led to formation of 3-amino 3,6-dideoxy- and 4-amino 4,6-dideoxyhexoses, respectively. Acetylation with acetyl coenzyme A yields the naturally-occurring N-acetyl amino sugar derivatives. 

Chelating bis(phenoxy) amino-donor complexes, with Zr(IV)... 

A transaminase patented by Celgene Corporation (Warren, NJ), called an co-aminotransferase [(co-AT)E.C. 2.6.1.18] does not require an a-amino acid as amino donor instead it requires a primary amine and hence has the ability to produce chiral amines.125 126 A similar co-AT from Vibrio fluvialis has been described for the production of chiral amines along with chiral alcohols when coupled with AdH or chiral amino acids when coupled with an a-amino acid aminotransferase.127130 Another co-AT, ornithine (lysine) aminotransferase (E.C. 2.6.1.68), has been described for the preparation of a chiral pharmaceutical intermediate used in the synthesis of Omapatrilat, a vasopep-tidase inhibitor developed by Bristol-Myers Squibb, as well as the UAA A1 -piperidinc-6-carboxylic acid.131-132... 

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