Enzyme amino acid transaminases

There is a further possibility for conversion of methionine to MT. Transaminase enzymes can convert methionine to 4-methylthio-2-oxobutanoate (also, a-keto-y-methylthiobutyrate). One such enzyme is aromatic-amino-acid transaminase, EC 2.6.1.57, for which L-methionine is, albeit, less efficiently, a substrate (Equation 5) ... 

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 ... 

Turning to l-AAO, Pantaleone s industrial research group have reported" on the properties and use of an l-AAO from Proteus myxofaciens, overexpressed in Escherichia coli This l-AAO, unusually, appears not to produce H2O2 in the catalytic reaction, thus making the addition of catalase unnecessary. The enzyme has a broad specificity, with a preference for nonpolar amino acids. This l-AAO was used in conjunction with a D-amino acid transaminase (d-AAT) and an alanine racemase (AR) to allow an efficient conversion of L-amino acid in to D-amino acid (Scheme 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... 

Another useful transaminase, D-amino acid transaminase (DAT) (E.C. 2.6.1.21), has been the subject of much study.53 133 134 This enzyme catalyzes the reaction using a D-amino acid donor, either alanine, aspartate, or glutamate (Scheme 19.20), to produce another D-amino acid. 

Amino acids NAD(P)H Pyridoxamine 5 -phosphate Pyruvate Amines Pyridoxal 5-phosphate dependent enzymes Dehydrogenases Transaminases Pyridoxal 5-phosphate dependent enzymes Amino acid decarboxylases... 

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... 

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). 

L-Amino acid transaminases are ubiquitous in nature and are involved, be it directly or indirectly, in the biosynthesis of most natural amino acids. All three common types of the enzyme, aspartate, aromatic, and branched chain transaminases require pyridoxal 5 -phosphate as cofactor, covalently bound to the enzyme through the formation of a Schiff base with the e-amino group of a lysine side chain. The reaction mechanism is well understood, with the enzyme shuttling between pyridoxal and pyridoxamine forms [39]. With broad substrate specificity and no requirement for external cofactor regeneration, transaminases have appropriate characteristics to function as commercial biocatalysts. The overall transformation is comprised of the transfer of an amino group from a donor, usually aspartic or glutamic acids, to an a-keto acid (Scheme 15). In most cases, the equilibrium constant is approximately 1. 

We have previously described the isolation and cloning of genes encoding microbial L-amino acid transaminases [40], and the use of site-directed mutagenesis to enhance enzyme function [41]. The reversibility of the reaction has frequently been considered a drawback of transaminase commercial application because it results in reduced yields and complicates product isolation. Through molecular cloning we have constructed bacterial strains that effectively eliminate this concern by removal of the keto acid by-product, and thereby significantly increase the reaction yield. 

The Stickland reaction (47) has received much attention as a possible route to chiral acetate due to the availability of chiral glycine (48). In the Stickland reaction two moles of glycine and one mole of d-alanine are converted quantitatively into three moles of acetate, three moles of ammonia, and one mole of C02 by the organism Clostridium sticklandii. The presence of amino acid transaminase in the intact organisms leads to extensive hydrogen exchange although in the purified enzyme the replacement of NH2 by H occurs stereospecifically with inversion (49, 50). Unfortunately, the rates of conversion with the purified enzyme are too low to be synthetically useful. 

Transaminases are generally not considered to be enzymes catalyzing redox reactions, which is obvious considering the meaning of the E. C. code for transferases (E. C. 2.6.1.x = transferring amino groups). Nevertheless, the exchange of an amino functionality between an amino acid and an a-keto acid implies the oxidation of the amino acid. Transaminases are described elsewhere in this book (Chapter 12). 

Pyridoxal phosphate is a co-enzyme for numerous enzymes, notably amino acid decarboxylases, amino acid transaminases, histaminase and probably diamine oxidase Ais.iw. As most of the evidence on which the mechanism of action of pyridoxal-dependent enzymes is based has been obtained from studies of the non-enzymic interaction of pyridoxal with amino acids, these non-enzymic reactions will be considered first in some detail. 

Scheme 12.16. A representation of the final stages in the biosynthesis of isoleucine (He, I). In the late stages shown here, the migration of the ethyl group is accompanied by the reduction of the carbonyl generated in that migration (the enzyme is a reductoisomerase (EC 1.1.1.86). Dehydration to an enol (a dihydroxy acid dehydratase, EC4.2.1.9), tautomeriza-tion to the corresponding ketone, and a final transamination ( branched-chain amino acid transaminase with pyridoxal as a cofactor) from glutamate (Glu,E) produces isoleuciue (He, I). EC numbers and some graphic materials provided in this scheme have been taken from appropriate links in a URL starting with http //www.chem.qmul.ac.uk/iubmb/enzyme/.

Thus, for phenylalanine (Phe, F), decarboxylation and dehydration (prephenate dehydratase, EC 4.2.1.51) to phenylpyruvate is followed by transamination with either of the enzymes tyrosine transaminase (EC 2.6.1.5) or aromatic amino acid transaminase (EC 2.6.1.57). Both of these use pyridoxal as cofactor and derive the nitrogen for the amino function from glutamate (Glu, E). 

An aromahc L-amino acid transaminase from Enterobacter sp. with unknown structure and no substrate specificity toward aromahc amino acid analogs was used to synthesize the L-phenylalanine analog L-diphenylalanine. Based on the interaction of fhe substrafe wifh the achve site of fhe enzyme, sife-directed mutagenesis can alter the substrate specificity of the transaminase toward substrates with bulky side chains [118]. 

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 ... 

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. 

A group of enzymes which is particularly important in amino acid metabolism in the liver (and also in muscle) is the transaminases, (also called aminotransferases). These are vitamin B6 (pyridoxine) dependent enzymes which transfer an amino group from an amino acid to an oxo (keto) acid, thus ... 

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