Amino adds aminotransferases

A racemase brings about inversion of the relatively cheap (L)-isomers of alanine or aspartic acid, but not of (D)-phenylalanine. Only (L)-phenylalanine is deaminated by an (L)-amino acid deaminase, whereas (D)-phenylalanme is not. The latter is generated by ammonia transfer from (D)-alanine or (D)-aspar-tic acid with a (D)-amino acid aminotransferase. The equilibria are moved in favour of the product, either by the metabolism of pyruvic acid or oxosuccinic acid. Since (L)-amino acid deaminases, like (D)-amino add aminotransferases, are non-specific, they also permit the preparation of a variety of other (D)-amino acids. [58]... 

Figure 8.11 Five near-equilibrium reactions involved in transamination of five different amino adds. Three enzymes are involved in these reactions (1) alanine aminotransferase (2) aspartate aminotransferase (3) branched-chain amino acid aminotransferase, i.e. one enzyme catalyses the three reactions. (The branched-chain amino acids are essential.)...

J. D. Rozzell, Immobilized aminotransferases for amino add production, Meth. Enzymol. 1987, 336, 479 197. 

Bakkelund AH, Fonnum F, Paulsen RE (1993) Evidence using in vivo microdialysis that aminotransferase activities are important in the regulation of the pools of transmitter amino adds. Neurochem Res 18 411-415. 

The conversion of amino acids to keto acids is usually catalyzed by enzymes called aiuinotransferases. Some aminotransferases can recognize a variety of different amino adds as substrates others are more specific in their action. Aminotransferases use vitamin as a cofactor, and the in the figures indicates that the enzyme is a vitamin B -requiring enzyme. Cofactors are small molecules that bind to specific enzymes and participate in the chemistry of cataly.sis. Some... 

The results suggested that a group from fhe profein abstracfs a proton from fhe a posihon and transfers if on fhe same side of fhe n system (suprafacial transfer), adding it to the si face of fhe C=N group as shown in Fig. 14-8. Later, fhe same stereospecific proton transfer was demon-sfrafed for the PLP present in the holoen-zyme. Not surprisingly, the D-amino acid aminotransferase adds the proton to the re face of the C = N group. 

A simple procedure was established for the synthesis of various D-amino adds by means of four types of thermostable enzymes alanine racemase, D-amino acid aminotransferase 49, 501, L-alanine dehydrogenase 51, and formate dehydrogenase (Fig. 17-4) 171. The commercial preparation of formate dehydrogenase from Candida boidinii used by Wichmanri et al. 38 is not sufficiently stable. However, Galkin et al.1521 doned and expressed the gene of thermostable formate dehydrogenase in E. coli. 

The experiments conclusively prove that the addition of hydrogen to C-4 of the coenzyme occurs at the Si face of the Schiff base. Evidence has already been provided for the syn nature of the tautomeric process in the reaction catalysed by pyridoxamine-pyruvate aminotransferase [107]. If the same precedent is extended to aspartate aminotransferase it then follows that the bond to C that is formed and broken in this case must also be located on the Si face at C-4, in the catalytic complex, as shown in structure 2 (Fig. 53). In other words, the alternative arrangement for syn proton transfer shown in 1 (Fig. 53) is precluded by these experiments. The direction of hydrogen addition to C-4 of the coenzyme in the half-reaction has also been studied using several other L-amino add requiring aminotransferases and in every case the medium hydrogen was shown to add to the Si face at C-4 (Table 5). These experiments have led to the generaUsed view that in B -dependent reactions... 

When the reaction occiurs as shown in Eqn. 3, the enzyme nomenclature system currently in vogue uses only the name of the unique amino add to spedfy an aminotransferase, e.g. aspartate aminotransferase, alanine aminotransferase, etc. etc. 

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 -hydrogen of the amino add is not a participant [37-40]. 

Now, with aspartate (Asp, D) in hand, for example, it should be clear that transamination will allow production of both essential and nonessential amino adds. Alanine (Ala, A), as shown in Scheme 12.2, can be formed when pyruvate (derived, e.g., from phosphoenolpyruvate via 2-phosphoglycerate, as shown inter alia Scheme 11.25) undergoes transamination from aspartate (Asp, D) (produced as in Equation 12.3 from fumarate). Fumarate, it will be remembered (Scheme 11.89), is produced in the tricarboxylic acid cycle. So, as shown in Scheme 12.2, the aminotransferase cofactor, pyridoxal, serves to deaminate aspartate (Asp, D) to produce oxaloacetate. The amino group is then transferred to pyruvate to yield alanine (Ala, A) and the pyridoxal cofactor is regenerated. ... 

Stirling, D., CoDins, A.N., Sheldrake, G.N., and Crosby, J. (eds) (1992) The use of aminotransferases for the production of chiral amino adds and amines, in Chirality in Industry, John Whey Sons, Ltd, Chichester, UK, 209-222. n Shin, J. and Kim, B. (1997) Kinetic resolution of (S)-mefhylbenzylamine with (o-ammotransferase screened from soil microorganisms application of a bi-phasic system to overcome product... 

PAL, phenylalanine ammonia lyase CA4H, cinnamic acid 4-hydroxylase CPR, Cytochrome P450 reductase 4CL, 4-coumaroyl-CoA ligase CA3H, coumaric add 3-hydroxylase COMT, caffeicacid O-methyltransferase SAM, S-adenosyl-methionine HCHL, hydroxycinnamoyl-CoA pAMT, putative aminotransferase AA, amino add KA, Keto add... 

Fig. 2.4 Phylogenetic tree based on amino add sequences of aminotransferases was constructed according to the method used to construct the tree in Fig. 2.3...

In nature, aminotransferases participate in a number of metabolic pathways [4[. They catalyze the transfer of an amino group originating from an amino acid donor to a 2-ketoacid acceptor by a simple mechanism. First, an amino group from the donor is transferred to the cofactor pyridoxal phosphate with formation of a 2-keto add and an enzyme-bound pyridoxamine phosphate intermediate. Second, this intermediate transfers the amino group to the 2-keto add acceptor. The readion is reversible, shows ping-pong kinetics, and has been used industrially in the production ofamino acids [69]. It can be driven in one direction by the appropriate choice of conditions (e.g. substrate concentration). Some of the aminotransferases accept simple amines instead of amino acids as amine donors, and highly enantioselective cases have been reported [70]. 

Nucleophilic groups from enzymes can add to double bonds, e.g., in an aminoacrylate Schiff base, or to multiple bonds present in die inhibitor. An example is y-vinyl y-aminobutyrate (4-amino-5-hexenoic acid), another inhibitor of brain y-aminobutyrate aminotransferase which is a useful anticonvulsant drug. 

Free amino acids are further catabolized into several volatile flavor compounds. However, the pathways involved are not fully known. A detailed summary of the various studies on the role of the catabolism of amino acids in cheese flavor development was published by Curtin and McSweeney (2004). Two major pathways have been suggested (1) aminotransferase or lyase activity and (2) deamination or decarboxylation. Aminotransferase activity results in the formation of a-ketoacids and glutamic acid. The a-ketoacids are further degraded to flavor compounds such as hydroxy acids, aldehydes, and carboxylic acids. a-Ketoacids from methionine, branched-chain amino acids (leucine, isoleucine, and valine), or aromatic amino acids (phenylalanine, tyrosine, and tryptophan) serve as the precursors to volatile flavor compounds (Yvon and Rijnen, 2001). Volatile sulfur compounds are primarily formed from methionine. Methanethiol, which at low concentrations, contributes to the characteristic flavor of Cheddar cheese, is formed from the catabolism of methionine (Curtin and McSweeney, 2004 Weimer et al., 1999). Furthermore, bacterial lyases also metabolize methionine to a-ketobutyrate, methanethiol, and ammonia (Tanaka et al., 1985). On catabolism by aminotransferase, aromatic amino acids yield volatile flavor compounds such as benzalde-hyde, phenylacetate, phenylethanol, phenyllactate, etc. Deamination reactions also result in a-ketoacids and ammonia, which add to the flavor of... 

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