Phenylpyruvate transaminase

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. 

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 19-1. Pathways for the metabolic disposal of phenylalanine. There are two competitive pathways for the disposal of phenylalanine. One pathway involves a transaminase enzyme phenylpyruvate, while the first step in the second pathway requires phenylalanine to be initially converted to tyrosine. Continued metabolism of the phenylpyruvate produced by the first pathway leads to products that cannot be further metabolized, while tyrosine can be converted into citric acid cycle intermediates. Glu, glutamate aKG CoASH, coenzyme A BH4, tetrahydrobiopterin TPP, thiamine pyrophosphate.

Reaction time (min) Transaminase alone Phenylpyruvate (mM) Transaminase Oxaloacetate decarboxylase Phenylpyruvate (mM)... 

The effectiveness of decarboxylation in driving the reaction to completion was demonstrated in a coupled enzymatic process by using phenylpyruvate as the starting 2-keto add. In this experiment, phenylpyruvate sodium salt and L-aspartate were incubated with E. coli broad-range transaminase at room temperature and pH... 

Transaminase model I could make the reaction rate of indole pyruvic acid transferring into indole alanine being 200-fold faster than PM, which makes the enantiomer surplus percentage of L-Trp generated from indole pyruvic acid and L-Ala generated from phenylpyruvic acid reach up to 12% and 67%, respectively. The reaction had obvious optical induction [17]. 

Depending on the substrate preference of the employed transaminase, the following couples of sacrificial amine donor/keto acceptors were used, which are often derived from the a-aminoacid pool (such as alanine/pyruvate, phenylalanine/ phenylpyruvate, glutamic acid/a-ketoglutarate, aspartic acid/a-ketosuccinate) or constitute simple amines/ketones, such as 2-propylamine/acetone and 2-butyl-amine/2-butanone. It should be kept in mind that the absolute configuration of a chiral amine-donor has to match the stereospecificity of the co-TA in order to be accepted. 

The accumulating metabolite is metabolized through alternative pathways, mainly the transaminase reaction. Consequently, imidazole pyruvic acid accumulates in the urine. The imidazole pyruvic, like phenylpyruvic, acid reacts with ferric chloride to yield a blue compound. As a result, the diaper test does not distinguish between phenylketonuria and histidinemia. Yet the diagnosis is of considerable importance because histidinemia is a much more benign disease. Furthermore, histidinemia is not alleviated by withdrawal of phenylalanine from the diet. 

A great deal of early research in the field established the foundations that now govern the concept of enzyme mimicry using MIPs, including the importance of structure-function relationships in determining binding and catalytic activity. Nicholls etal. prepared and evaluated an MIP transaminase mimic for the reaction of phenylpyruvic acid and pyridoxamine (Scheme 22). ... 

Thus, for phenylalanine (Phe, F), decarboxylation and dehydration (prephenate dehydratase, EC 4.2.1.51) to phenylpyruvate is followed by transamination with either of the enzymes tyrosine transaminase (EC 2.6.1.5) or aromatic amino acid transaminase (EC 2.6.1.57). Both of these use pyridoxal as cofactor and derive the nitrogen for the amino function from glutamate (Glu, E). 

The a-keto adds of the above amino acids (except methionine) and also o-ketoglutarate were effective substrates for transamination. Study of the kinetic properties of this transaminase preparation yielded the following results optimum activity at pH 8 K, values phenylpyruvate, 1.2 X M phenylalanine, 13 X 10 Af -keto-j8-methylvalerate, 10 X 10 M isoleucine, 59 X 10 Af a-ketoisovalerate, 15 X 10 Af valine 111 X 10 Af oE-ketoglutarate, 24 X 10 M. Determination of the equilibrium constant for a-keto-/9-methylvalerate conversion to isoleucine with phenylalanine as amino group donor, yielded an average value of approximately 0.50. 

---