Aminotransferases transformation

Chiral amines can also be produced using aminotransferases, either by kinetic resolution of the racemic amine or by asymmetric synthesis from the corresponding prochiral ketone. The reaction involves the transfer of an amino group, a proton and two electrons from a primary amine to a ketone, and proceeds via an intermediate imine adduct. A variety of chiral amines can be obtained with high to very high ee-values. Several transformations have been developed and can be carried out on a 100-kg scale [94]. 

Previously, AAT had been transformed into an L-tyrosine aminotransferase (TAT) by site-specific mutation of up to six amino acid residues lining the active site of wild-type AAT. The hextuple AAT-mutant achieved kinetic data towards the transamination of aromatic substrates such as i-phenylalanine within an order of magnitude of wild-type TAT (Onuffer, 1995). 

The studies culminating in the proposal of the mechanism of action of aminotransferases as outlined in Fig. 10, include the early observation that the transformation occurs without intermediate formation of ammonia [35] and with the acquisition of one proton from the solvent which is located at C in the new amino acid [36]. It was also shown that the 8-hydrogen of the amino acid is not a participant [37-40]. 

A bacterial aminotransferase [46] promotes a decarboxylative transamination reaction with a-aminoisobutyrate in the presence of pyruvate. The reaction occurs via the sequence of Fig. 12 involving an initial cleavage of the Q-COjH bond in the substrate pyridoxal-P Schiff base complex (Fig. 12, 1) followed by reprotonation at C-4 of the coenzyme to give the pyridoxamine-P-enzyme complex (Fig. 12, 4) that participates in the transamination of pyruvate. However, the enzyme will also transform L-alanine at a significant rate by a half-transamination reaction into pyruvate, thereby implying that it is now the C -H bond of the amino acid that is first broken. 

The pattern recorded above raises the question whether the change of face at C-4 to the solvent side is a mandatory requirement in the transformation of binary into ternary complexes in pyridoxal-P-dependent reactions. That this may be so was the view beginning to prevail until a timely reminder, or perhaps an undue caution, came from a more recent report by Zito and Martinez-Carrion [93]. As has already been cited, these workers repeated the earlier experiments of the Zurich School on aspartate aminotransferase confirming the Re face hydride attack at C-4 in the binary complex. However, aspartate aminotransferase carbamylated at the active site Lys-258 was used to produce the substrate-coenzyme Schiff base linkage in the ternary complex. Since the modified enzyme catalysed the half-transamination reaction ... 

This reaction, of course, produces racemic amines. But nature transforms this simple reaction into an enantioselective and reversible one that is beautiful in its simplicity. The reagents are a pair of substituted pyridines called pyridoxamine and pyridoxal, and the enzyme is an aminotransferase. 

Some enzyme reactions can be studied colorimetrically when either the substrate or product can be converted chemically to a coloured product suitable for measurement in a u.v. or visible light spectrophotometer. In the case of alanine aminotransferase, the pyruvate formed in the reaction can be converted to pyruvate-2,4-dinitrophenylhydrazone by the addition of 2,4-dinitrophenylhydrazine (DNP). Addition of sodium hydroxide yields a product with an absorption maximum at 505 nm. Other examples of colorimetric procedures will be found in the last section. Colorimetric procedures are used for enzyme assays in the sampling mode, whereby samples of the reaction mixture are analysed at certain fixed times after starting the reaction. Graphs depicting the reaction rate must then be constructed by plotting amount of substrate transformed against time. 

One of the known mechanisms of biosynthesis is shown in the next scheme. The enzyme aminotransferase transfers the amino group from the precursor amino acid to the a-ketoacid, which is rearranged into the product amino acid. In this process, the precursor amino acid is transformed into the corresponding a-ketoacid. It must be pointed out that besides transferring the amino group, the enzyme aminotransferase also preserves the stereochemistry the L-precursor amino acid leads to the formation of the L-product aminoacid. This mechanism presented below is for the biosynthesis of L-glutamic acid. 

Amino acid decarboxylases transform amino acids to the corresponding amines. Aminotransferases catalyze a binary mechanism illustrated by the following equations ... 

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