A-transaminases

The high toxicity of AOA is due to its very high efficiency as a transaminase inhibitor (K =0.45 pM) as compared to its efficacy as a PAL inhibitor (K. = 120 pM) (48), making it impossible to effectively inhibit PAL iti vivo without also greatly inhibiting amino acid metabolism. Other pyridoxyl phosphate-requiring enzymes, such as ACC synthase (an enzyme involved in ethylene production) (49), are also more sensitive to AOA than to AOPP. 

Glutamate can then participate in the formation of other amino acids via the process called transamination. Transamination is the exchange of the amino group from an amino acid to a keto acid, and provides the most common process for the introduction of nitrogen into amino acids, and for the removal of nitrogen from them. The reaction is catalysed by a transaminase enzyme, and the coenzyme pyridoxal phosphate (PLP) is required. 

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 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... 

One of the earliest published attempts to create antibodies with catalytic activity had as its goal the generation of a transaminase. Raso and Stollar prepared V-(5-phosphopyridoxyl)-3 -amino-L-tyrosine 154 as a mimic of the Schiff s base intermediate that is formed during the pyridoxal-dependent transamination of tyrosine and showed that it was a site-directed inhibitor of the enzymes tyrosine transaminase and tyrosine decarboxylase.132 Partially purified polyclonal antibodies, elicited against y-globulin conjugates of the hapten, recognized both the... 

Quinolizidine Alkaloids.—Important new information (cf. Vol. 11, p. 4) has been obtained on the biosynthesis of quinolizidine alkaloids such as lupanine (27) in experiments with enzyme preparations from Lupinus polyphyllus cell suspension cultures26 and with chloroplasts.27 These alkaloids are formed from three molecules of lysine by way of cadaverine (25),1,2 and the enzymic evidence26,27 is that conversion of cadaverine into these alkaloids occurs without release of intermediates until 17-oxosparteine (26) is generated the enzyme is a transaminase and not a diamine oxidase. 

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.

Another case of the use of a single round of mutagenesis to improve the enantioselectivity of an enzyme (which therefore is not an evolutionary process) concerns the enantioselectivity of a transaminase. Transaminases are enzymes which catalyze the conversion of ketones to the corresponding primary amines, the ee values often being >95 % [85]. However, in one case an ee of only 65 % was observed [86], A single library of variants was produced which contained an enzyme showing 98 % ee. Unfortunately... 

Researchers have Isolated and characterized a transaminase from . coll K-12, specific for production of L-phosphInothrIcIn (I2g). Further work has resulted In cloning, characterization, and overexpression of the gene coding for the L-phosphlnothrlcIn-speclflc transaminase from . coll K-12. These results greatly facilitate biotechnological production of L-phosphInothrIcIn In an enzyme reactor. 

Figure 24.9. Amino Acid Biosynthesis by Transamination. Within a transaminase, the internal aldimine is converted into pyridoxamine phosphate (PMP) by reaction with glutamate. PMP then reacts with an a-ketoacid to generate a ketimine. This intermediate is converted into a quinonoid intermediate, which in turn yields an external aldimine. The aldimine is cleaved to release the newly formed amino acid to complete the cycle.

Figure 24.10. Stereochemistry of Proton Addition. In a transaminase active site, the addition of a proton from the lysine residue to the bottom face of the quinonoid intermediate determines the 1 configuration of the amino acid product. The conserved arginine residue interacts with the a-carboxylate group and helps establish the appropriate geometry of the quinonoid intermediate.

C) alanine may be produced from serine by the action of a dehydratase followed by the action of a transaminase... 

INDOLMYCIN (20) is formed from pyruvate, and two enzymes active in initial stages of Its biosynthesis have been studied. They are a transaminase and aC-methyltransferase. The hypothetical route to indolmycin is by indole pyruvate, 3-methyl-indolepyruvate, indolmycenic acid (reduced alpha oxo group) and finally indolmycin which probably takes its amidine group from an arginine molecule 79. The closely related [pyrrolo (1,4) benzodiazepines] 80>81,82 antitumor antibiotics, anthramycin, tomaymycin and sibiromycin are formed from tryptophan (via the kynurenine pathway ), tyrosine and methionine-derived methyl groups 80.si.sz. 

Wiss (937, 938) fractionated crude liver extracts to give a kynureninase fraction which would form anthranilic acid, but not kynurenic acid, and a transaminase fraction which would not form anthranilic acid, but formed kynurenic acid provided an a-keto acid was present. o-Aminobenzoyl-pyruvic acid, the keto acid corresponding to kynurenine, is known to cyclize spontaneously to kynurenic acid (622), and the absence of ammonia production and requirement for an a-keto acid (c/. also 434) suggests that... 

The aminotransferase, or transaminase class of enzymes, are ubiquitous, PLP-requiring enzymes that have been used extensively to prepare natural L-amino acids [84,85]. They catalyze the general reaction shown in Scheme 15, where an amino group from one L-amino acid is transferred to an a-keto acid to produce a new L-amino acid and the respective a-keto acid. Those enzymes most commonly used have been cloned, overexpressed, and generally used as whole cell or immobilized preparations. These include the following branched chain aminotransferase (SCAT) (EC 2.6.1.42), aspartate aminotransferase (AAT) (EC 2.6.1.1), and tyrosine aminotransferase (TAT) (EC 2.6.1.5). A transaminase patented by Celgene Corporation (Warren. NJ), called an co-aminotransferase, does not require an a-amino acid as amino donor and hence is used to produce chiral amines [86,87]. Another useful transaminase, n-amino acid transaminase (DAT) (EC 2.6.1.21), has been the subject of much study [37,88,89]. This enzyme catalyzes the reaction using a n-amino acid donor, either alanine or aspartate (Scheme 16). 

This removal of the reaction by-product has been achieved through the use of aspartic acid as the amino donor (Scheme 16). The amine group transfer results in the fonnation of oxaloacetate (7), an unstable compound that decarboxylates under the reaction conditions to afford pyruvate (8). As 8 is still an a-keto acid, and is a substrate for a transaminase reaction that results in the production of alanine, another enzyme is used to dimerize the pyruvate. The product of this reaction is acetolactate (9), which, in turn, spontaneously undergoes decarboxylation to result in the overall formation of acetoin (10) as the final by-product. Acetoin is simple to remove and does not participate in any further reactions. Thus, the equilibrium is driven to provide the desired unnatural amino acid that makes the isolation straightforward. 

Figure 14.22 Resolution of racemic chiral amines using a transaminase from 6. megaterium.

The evolution of a transaminase from Arthrohacter citreus to a thermostable transaminase with increased specific activity and decreased inhibition by the amine product was accomplished using error prone polymerase chain reaction (PCR) [64] The reaction of substituted tetralone 75 and isopropylamine to produce substituted (S) aminotetralin 76 was carried out at greater than 50 °C to facilitate the removal of the acetone by product and drive reaction equilibrium (Figure 14.43). 

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]. 

Another example of a coupled enzyme reaction demonstrates the versatility of the transaminase system in biocatalysis. Using a racemic d,L-amino acid mixture as the starting material, the enzyme D-amino acid oxidase from Trigonopsis mriabilis will convert the D-amino acid in the mixture selectively into the corresponding 2-keto acid. The L-amino acid of the d,l- pair is neither a substrate nor an inhibitor of d-amino acid oxidase. If a transaminase is present in the same reaction mixture, the 2-keto acid can be transaminated in the presence of L-aspartate to the corresponding L-amino acid. The entire reaction can be driven to completion as described previously by decarboxylation of the oxaloacetate. Thus, in a single pot, racemic d,l-amino acids can be convened directly into optically active L-amino acids (Fig. 12.7-11). 

Transferases are enzymes that catalyze the transfer of functional groups from one molecule to another. For example, a transaminase catalyzes the transfer of an amino functional group, and a kinase catalyzes the transfer of a phosphate group. Kinases play a major role in energy-harvesting processes involving ATP. In the adrenal glands, norepinephrine is converted to epinephrine by the enzyme phenylethanolamine-N-methyltransferase (PNMT), a transmethylase. 

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