Transaminase

Amino transferases (transaminases) catalyze the PLP-dependent reversible transfer of an amino and keto group between an amino acid donor and a keto acid acceptor yielding new amino acid and keto acid product, according to the equation 

When the amino donor has the L-configuration and an L-amino acid transaminase is used, the product amino acid will have an L-configuration and when the amino donor has the D-configuration and a D-amino transaminase is used, the product amino acid will have a D-configuration. The equilibrium constant for the reaction is typically close to unity. 

L-Amino transaminase activities are ubiquitous in Nature since they are involved in the biosynthesis of most natural amino acids. On the other hand, D-amino transaminase activities have been identified in bacteria, mostly in the Bacillus strains and are involved in the production of D-amino acids for the peptidoglycan layer of the cell wall. The mechanism is weU established, with PLP shuttHng 

Transaminases possess many features appropriate for effident biocatalysts, such as high turnover numbers and no requirement for external recycling of the co-factor. Because of the wide substrate tolerance of many amino transferases such as tyrosine amino transferase and branched-chain amino transferases from E. coU, these enzymes have been largely employed in the enantiospecific preparation of non-proteinogenic amino acids. These include straight-chain alkyl, diadd, branched-chain, aromatic, and bifunctional amino adds [65]. 

The most commonly used L-amino transferase activities for L-amino acid synthesis are the ones from E. coli, which can be used in whole cell or immobilized systems. They include the following. First, aspartate amino transferase (EC 2.6.1.1), the 

In the following discussion of more recent results on the stereochemistry of PLP enzyme reactions, some of the newer data will be analyzed in terms of these early concepts. 

Aminotransferases (transaminases) catalyze the reversible interconversions of pairs of a-amino and a-keto acids or of terminal primary amines and the corresponding aldehydes by a shuttle mechanism in which the enzyme alternates between its PLP form and the corresponding PMP form. In the first half-reaction the PLP form of the enzyme binds the amino acid (or amine) and forms the coenzyme-substrate Schiff s base. Cleavage of the C-a—H bond is then followed by protonation at C-4. Hydrolysis of the resulting ketimine then gives a keto acid (or aldehyde), leaving the enzyme in the PMP form. The latter is recycled to the PLP form by condensation with an a-keto acid, deprotonation at C-4, protonation at C-a and transaldimina-tion to release the a-amino acid formed. 

Knowledge of four of these five parameters is sufficient to completely describe the system. In the case of a-amino acids the configuration at C-a is usually known a 

For two transaminases the remaining unknown stereochemical parameter was determined by demonstrating an internal transfer of tritium (dialkyl amino acid transaminase) [28] or deuterium (pyridoxamine-pyruvate transaminase) [27] from the a-position of the substrate L-alanine to C-4 of the cofactor. Internal hydrogen transfer from the a-position of the substrate amino acid to C-4 of PLP has also been demonstrated for two of the abortive transamination reactions, those catalyzed by tryptophan synthase fi2 protein [32] and by aspartate-/8-decarboxylase [31]. In addition, the same phenomenon must occur in alanine transaminase, as deduced from the observation that the enzyme catalyzes exchange of the /8-hydrogens of 

L-aspartate. They observed that tritium was introduced at C-4 of the cofactor from the Re face to the extent of over 90%. Thus the exposed face of the coenzyme-substrate complex in the modified enzyme is the Re face as in the holoenzyme rather than the Si face as in the complex of the normal enzyme with L-aspartate. Yet this modified enzyme is still able to undergo the half-transamination reaction with conversion of active site bound PLP to PMP [45] and does so with stereospecific protonation of the cofactor from the Si face [46]. According to this result, a change in the exposed face of the cofactor upon transaldimination or a conformation in which the Si face of C-4 is exposed in the coenzyme-substrate complex are not requirements for catalytic activity. Likewise, the face on which reactions take place in the catalytic process is not necessarily the face that is most accessible to external reagents. 

Celgene developed transaminase technology for the enantioselective conversion of chiral amines to ketones [25]. Low molecular weight aldehydes, such as propional dehyde, were used as the amine group acceptor. This process has been used on a 

A significant drawback of the transaminase approach is the need for a stoichio 

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ANALYTICALTffiTHODS - HYPHENATED INSTRUTffiNTS] (Vol 2) Leucine a-ketoglutarate transaminase... 

Transaminases Transamination Transannular peroxide Transcat process Transcobalamin II Transcortin... 

Deamination, Transamination. Two kiads of deamination that have been observed are hydrolytic, eg, the conversion of L-tyrosiae to 4-hydroxyphenyUactic acid ia 90% yield (86), and oxidative (12,87,88), eg, isoguanine to xanthine and formycia A to formycia B. Transaminases have been developed as biocatalysts for the synthetic production of chiral amines and the resolution of racemic amines (89). The reaction possibiUties are illustrated for the stereospecific synthesis of (T)-a-phenylethylamine [98-84-0] (ee of 99%) (40) from (41) by an (5)-aminotransferase or by the resolution of the racemic amine (42) by an (R)-aminotransferase. 

L-3-metliylvaline 3, 3-dimethyl-2-oxobutyric acid + Asp transaminase Cryptococcus leurentii 197... 

L-methionine DL-methionine + Asp D-amino acid oxidase + transaminase Trigonopsis variabilis ... 

L-2-ainino-4-plienylbuty 2-keto-4-phenylbutyric acid transaminase ... 

Glutamic acid dehydrogenase is widely distributed in microorganisms and higher plants as a catalyst in the synthesis of L-glutamic acid from a-ketoglutaric acid and free ammonia. Transaminase is contained in a wide variety of microorganisms. 

Alcohol dehydrogenase (5) and leucine a-ketoglutarate transaminase (33,34) contribute to the development of aroma during black tea manufacturing. Polyphenol oxidase and peroxidase are essential to the formation of polyphenols unique to fermented teas. 

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 ... 

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). 

Pyridoxamine phosphate serves as a coenzyme of transaminases, e.g., lysyl oxidase (collagen biosynthesis), serine hydroxymethyl transferase (Cl-metabolism), S-aminolevulinate synthase (porphyrin biosynthesis), glycogen phosphoiylase (mobilization of glycogen), aspartate aminotransferase (transamination), alanine aminotransferase (transamination), kynureninase (biosynthesis of niacin), glutamate decarboxylase (biosynthesis of GABA), tyrosine decarboxylase (biosynthesis of tyramine), serine dehydratase ((3-elimination), cystathionine 3-synthase (metabolism of methionine), and cystathionine y-lyase (y-elimination). 

Older adults are particularly susceptible to a potentially fatal hepatitis when taking isoniazd, especially if they consume alcohol on a regular basis. Two other antitubercular drugs rifampin and pyrazinamide, can cause liver dysfunction in the older adult. Careful observation and monitoring for signs of liver impairment are necessary (eg, increased serum aspartate transaminase, increased serum alanine transferase, increased serum bilirubin, and jaundice). 

A serious and potentially fatal adverse reaction to tolcapone ishepatic injury. Regular blood testing to monitor liver function is usually prescribed. The phys dan may order testing of serum transaminase levels at frequent intervals(eg, every 2 weeks for the first year and every 8 weeks thereafter). Treatment is discontinued if the ALT (SOFT) exceeds the upper normal limit or sgns or symptoms of liver failure develop. 

When administering tacrine, the nurse must monitor the patient for liver damage. This is best accomplished by monitoring alanine aminotransferase (AIT) levels. ALT is an enzyme found predominately in the liver. Disease or injury to the liver causes a release of tiiis enzyme into the bloodstream, resulting in elevated ALT levels, hi patients taking tacrine, ALT levels should be obtained weekly from at least week 4 to week 16 after die initiation of tiierapy. After week 16, transaminase levels are monitored every 3 months. 

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. 

SCOT serum glutamic oxaloacetic transaminase TSH thyroid-stimulating hormone... 

I 2 Directed Evolution as a Means to Engineer Enantioseleaive Enzymes (S)-transaminase... 

A more recent study focused on the directed evolution of the co-transaminase from Vibrio fiuvialis JS17, specifically with the aim to eliminate product inhibition by aliphatic ketones while maintaining high enantioselectivity. This was achieved by screening 85 000 clones produced by epPCR [72]. 

Activities of glutamate-pyruvate transaminase (SGPT, GPT) (EC 2.6.1.2), L-y -glutamyl-transferase (y-GT) (EC 2.3.2.2) and level of triglycerides (TG) in serum, as well as levels of glutathione (GSH) and malondialdehyde (MDA) in the liver were determined. 

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