Phenylalanine 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). 

With AOPP, the selectivity is reversed with the K for PAL being 1.4 nM and that for phenylalanine transaminase being 3 pM (48). Thus, AOPP can effectively block PAL activity in vivo without being strongly phytotoxic (e.g. 20, 32, 38). 

The liver is also the principal metabolic center for hydrophobic amino acids, and hence changes in plasma concentrations or metabolism of these molecules is a good measure of the functional capacity of the liver. Two of the commonly used aromatic amino acids are phenylalanine and tyrosine, which are primarily metabolized by cytosolic enzymes in the liver [1,114-117]. Hydroxylation of phenylalanine to tyrosine by phenylalanine hydroxylase is very efficient by the liver first pass effect. In normal functioning liver, conversion of tyrosine to 4-hy-droxyphenylpyruvate by tyrosine transaminase and subsequent biotransformation to homogentisic acidby 4-hydroxyphenylpyruvic acid dioxygenase liberates CO2 from the C-1 position of the parent amino acid (Fig. 5) [1,118]. Thus, the C-1 position of phenylalanine or tyrosine is typically labeled with and the expired C02 is proportional to the metabolic activity of liver cytosolic enzymes, which corresponds to functional hepatic reserve. Oral or intravenous administration of the amino acids is possible [115]. This method is amenable to the continuous hepatic function measurement approach by monitoring changes in the spectral properties of tyrosine pre- and post-administration of the marker. 

This enzyme [EC 2.6.1.1] (also known as transaminase A, glutamicioxaloacetic transaminase, and glutamic aspartic transaminase) catalyzes the reversible reaction of aspartate with a-ketoglutarate to produce oxaloace-tate and glutamate. Pyridoxal phosphate is a required cofactor. The enzyme has a relatively broad specificity, and tyrosine, phenylalanine, and tryptophan can all serve as substrates. 

This pyridoxal-phosphate-dependent enzyme [EC 2.6.1.5], also known as tyrosine transaminase, catalyzes the reaction of L-tyrosine with a-ketoglutarate (or, 2-oxoglutarate) to produce 4-hydroxyphenylpyruvate and L-glutamate. L-Phenylalanine can act as the substrate instead of tyrosine. In some systems, the mitochondrial enzyme may be identical with aspartate aminotransferase. 

However, when radioactive L-valyl-L-proline lactam was fed into cultures (94) an unexpected observation was made. Degradation studies revealed that it had been hydrolyzed prior to incorporation. This fact, together with the repeatedly postulated high transaminase activity of the system, could offer an explanation for an observation of Abe (95) who noticed that in cell-free systems from a strain of Elymus type of ergot fungus, L-phenylalanine-D-proline lactam was also incorporated into ergotamine. 

The fermentation methods used to prepare L-phenylalanine, threonine, lysine, and cysteine are discussed in detail in Chapter 3. The adaptation of these methods to prepare unnatural amino acids, such as the use of transaminases, is also discussed in that chapter. One of the large-scale amino acids, L-glumatic acid, which is often sold as its monosodium salt, is not covered because its preparation by fermentation is long established.3... 

The metabolic pathways of phenylalanine and tyrosine are identical, because the essential phenylalanine must be converted to tyrosine to become metabolized. Figure 20.22 illustrates this pathway, which is termed the liver pathway to distinguish it from those leading to catecholamine biosynthesis. It is localized in the cytosol, with the exception of tyrosine transaminase, which is also present in the mitochondria. 

Figure 20.22 Catabolism of phenylalanine and tyrosine. A indicates the lesion in classic phenylketonuria B indicates a tyrosinemia caused by tyrosine transaminase deficiency C indicates a tyrosinemia caused by p-hydroxyphenylpyruvate oxidase deficiency and the lesion in neonatal tyrosinemia D indicates alcaptonuria.

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.

Indolmydn.—Previous evidence on the biosynthesis of indolmycin (88) in Strepto-myces griseus cultures accords with the pathway shown in Scheme 4. The first two steps in the pathway have been carried out using cell-free extracts of 5. griseus - and recent work has led to the isolation of two enzymes which can effect these transformations. The first, tryptophan transaminase, catalysed the pyridoxal phosphate-dependent transamination of L-tryptophan, but not D-trptophan, and in common with some other microbial transaminases, a-ketoglutarate was an efficient amino-group acceptor. L-Phenylalanine, tyrosine, and 3-methyltryptophan (this compound inhibited enzyme function) also underwent transamination. 

All of the amino acids except lysine, threonine, proline, and hydroxyproline participate in transamination reactions. Transaminases exist for histidine, serine, phenylalanine, and methionine, but the major pathways of their metabolism do not involve transamination. Transamination of an amino group not at the a-position can also occur. Thus, transfer of 3-amino group of ornithine to a-ketoglutarate converts ornithine to glutamate-y-semialdehyde. 

Hepatic cytosolic tyrosine aminotransferase (tyrosine transaminase) deficiency produces tyrosinemia type II, an autosomal recessive trait marked by hypertyrosine-mia and tyrosinuria. Clinical manifestations may include corneal erosions and plaques, inflammation (from intracellular crystallization of tyrosine), and mental retardation. Low-tyrosine and low-phenylalanine diets are beneficial. 

A. The correct response is dihydropteridine reductase. This enzyme reduces dihydrobiopterin to tetrahydrobiopterin the obligate electron donor for phenylalanine hydroxylase. Tyrosinase is the first enzyme on the pathway to melanin. Dopamine hydroxylase and tyrosine transaminase are enzymes on other tyrosine metabohc tracts. Homogentisic acid oxidase is an enzyme on the pathway of tyrosine to fumarate and acetoacetate. 

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. 

An estimation of the amount of amino acid production and the production methods are shown ia Table 11. About 340,000 t/yr of L-glutamic acid, principally as its monosodium salt, are manufactured in the world, about 85% in the Asian area. The demand for DL-methionine and L-lysine as feed supplements varies considerably depending on such factors as the soybean harvest in the United States and the anchovy catch in Pern. Because of the actions of d-amino acid oxidase and L-amino acid transaminase in the animal body (156), the D-form of methionine is as equally nutritive as the L-forni, so that DL-methioiiine which is inexpensively produced by chemical synthesis is primarily used as a feed supplement. In the United States the methionine hydroxy analogue is partially used in place of methionine. The consumption of L-lysine has increased in recent years. The world consumption tripled from 35,000 t in 1982 to 100,000 t in 1987 (214). Current world consumption of L-tryptophan and L-threonine are several tens to hundreds of tons. The demand for L-phenylalanine as the raw material for the s57nthesis of aspartame has been increasing markedly. 

---