Leucine transaminase

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

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. 

The enzymes found in liver cells (Group I enzymes) include more than a dozen enzymes used in diagnostic laboratories, but those used most commonly are the transaminases (GOT and GPT), which continue to be the most widely used indicators of liver cell integrity. Enzymes found in the biliary cells (Group II) include alkaline phosphatase, glutamyl-transferase, leucine amniopeptidase and 3-nucleotidase. 

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 reactions are catalysed by enzymes known as aminotransferases (formerly known as transaminases). For the above reactions, they are (i) aspartate aminotransferase, (ii) alanine aminotransferase and (iii) leucine aminotransferase. Details of these reactions can be found in Appendix 8.4. 

This enzyme [EC 2.6.1.21], also known as D-aspartate aminotransferase, D-amino acid aminotransferase, and D-amino acid transaminase, catalyzes the reversible pyridoxal-phosphate-dependent reaction of D-alanine with a-ketoglutarate to yield pyruvate and D-glutamate. The enzyme will also utilize as substrates the D-stereoisomers of leucine, aspartate, glutamate, aminobutyrate, norva-hne, and asparagine. See o-Amino Acid Aminotransferase... 

This enzyme [EC 2.6.1.42], also referred to as transaminase B, catalyzes the reversible reaction of leucine with a-ketoglutarate (or, 2-oxoglutarate) to produce 4-methyl-2-oxopentanoate and glutamate. The pyridoxal-phosphate-dependent enzyme will also utilize isoleucine and valine as substrates. However, this enzyme is distinct from that of valine pyruvate aminotransferase [EC 2.6.1.66]. See also Leucine Aminotransferase... 

Recently, a lipase-catalysed kinetic resolution has been developed to produce L-tert-leucine via a lactone of its A-benzoyl derivative. There is also reputed to be another bioroute to L-terMeucine using transaminase technology. 

In all cases the keto acids seem to be formed by typical a-ketogluta-rate-linked, pyridoxal phosphate-dependent transaminases (EC 2.6.1.6, etc.) (9, 154, 156, 157). There has been little study of isolated, presumably specific enzymes in connection with flavors, although the leucine and alanine aminotransferases of tomato have been precipitated with (NH4)oS04 (164, 165). Transaminase activity in Saccharomyces cere-visiae has a pH optimum of 7.2 (154), and a-ketoglutarate is the only amino group recipient (154, 166). Only aspartate and amino acids with hydrophobic side chains are acted on (154). 

Branched Chain Amino Acids valine (val), leucine (leu), and isoleucine (ilu). The metabolism of each of these three amino acids begins with the same theme transaminase DH Complex foeta-oxidation. Due to the irreversible nature of the DH Complex all three are essential. 

Another example is provided by r>-tert-leucine (4) (Scheme 10), where an asymmetric approach cannot be used. This unnatural amino acid is one of the few that cannot be made by our current o-amino acid transaminase. While this enzymatic method is being researched, small amounts of material have been prepared by a resolution method [24]. 

A number of amino acids, like alanine, leucine, tyrosine, aspartic acid, cystein and arginine react with a-ketoacids and transfer their a-amino group to the a-carb-on of the a-keto acids. These reactions are catalysed by the enzyme called transaminase or aminotransferase. For example, transfer of the amino group of aspartic acid (14) to a-ketoglutaric acid (23) gives glutamic acid (16) and oxaloacetic acid (24). 

Biological systems can nse fermentative methods to produce amino acids. In some cases a metabolic pathway has been changed, bnt this is usually for natural amino acids, while in other cases an enzyme has been applied to a specihc transformation. An example is the use of transaminases (ronte g) to prodnce nnnatnral amino acids snch as 2-aminobutyrate and tcrt-leucine. 

Other examples of the nse of transaminases to synthesize unnatural amino acids have also been described in the literature, including L-tert-lencine (r-Tle) (9), L-2-amino-4-(hydroxymethylphos-phinyl)butanoic acid (phosphinothricin) and L-thienylalanines. Not all unnatural amino acids can be accessed by this technology. Althongh it works well for L-tert-leucine, D-tert-lencine remains elnsive. 

Commercial preparations of pig heart glutamate-oxaloacetate transaminase have been screened for their ability to transaminate various a-keto acids with l-[ N]glutamate (32). In addition to l-[ N]aspartate, enzyme preparations were able to catalyze the formation of labeled tyrosine, phenylalanine, leucine, and dihydroxyphenylalanine, as demonstrated by HPLC (17). However, these amino acids have not yet been obtained in radiopure form by this method. The -keto acid analogs of valine and tryptophan were not transaminated by the enzyme preparations. Glutamate-oxaloacetate transaminases obtained from several commercial sources have varying abilities to transaminate the -keto acid... 

There are several examples of d to l inversion of amino acids in the literature. D-Phenylalanine may have therapeutic properties in endogenous depression and is converted to L-phenylalanine in humans [145]. o-Leucine is inverted to the L-enantiomer in rats. When o-enantiomer is administered, about 30% of the enantiomer is converted to the L-enantiomer with a measurable inversion from l to o-enantiomer. As indicated in Fig. 13, D-leucine is inverted to the L-enantiomer by two steps. It is first oxidized to a-ketoisocarproate (KIC) by o-amino acid oxidase. This a-keto acid is then asymmetrically reaminated by transaminase to form L-leucine. In addition, KIC may be decarboxylated by branched-chain a-keto acid dehydrogenase, resulting in an irreversible loss of leucine (Fig. 13) [146]. D-Valine undergoes a similar two-step inversion process, and this can be antagonized by other amino acids such as o-leucine. The primary factor appears to be interference with the deamination process [147]. 

L-Leucine + 2-oxoglutarate - 2-oxo-isocaproate + L-glutamate This is a transaminase which reacts with 2-oxoglutarate of the citric acid cycle. 

FIGURE 12.1 The Ehrlich pathway exemplified for the conversion of the branched-chain amino acids L-valine, L-isoleucine, and L-leucine to the corresponding alcohols isobntanol, 2-methyl-1-bntanol, and 3-methyl-1-butanol. Adh, alcohol dehydrogenase KE)C, 2-ketoacid decarboxylase TA, transaminase. 

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