Transamination pyridoxal phosphate enzymes

Transamination Reactions of Other Pyridoxal Phosphate Enzymes Inaddition to theirmainreactions, anumberofpyridoxalphosphate-dependent enzymes also catalyze the half-reaction of transamination. Such enzymes include serine hydroxymethyltransferase (Section 10.3.1.1), several decarboxylases, and kynureninase (Section 8.3.3.2). 

The reactions of the Cu(II), Fe(III), and Al(III) chelates of Schiff bases formed by the condensation of pyridoxal with amino acids and peptides were found by Snell to have catalytic properties similar to those of the pyridoxal phosphate enzymes. A typical metal-catalyzed reaction of this type would be the transamination of pyridoxal and alanine according to... 

Fig. 38.4. Function of pyridoxal phosphate (PLP) in transamination reactions. The order in which the reactions occur is 1 to 4. Pyridoxal phosphate (enzyme-bound) reacts with amino acidj, forming a Schiff base (a carbon-nitrogen double bond). After a shift of the double bond, a-keto acidi is released through hydrolysis of the Schiff base, and pyridoxamine phosphate is produced. Pyridoxamine phosphate then forms a Schiff base with a-keto acidj. After the double bond shifts, amino acid2 is released through hydrolysis of the Schiff base and enzyme-bound pyridoxal phosphate is regenerated. The net result is that the amino group from amino acidj is transferred to amino acid2.

These reactions involve the activities of transaminases and decarboxylases (see p. 210), and over 50 pyridoxal phosphate-dependent enzymes have been identified. In transamination, pyridoxal phosphate accepts the a-amino group of the amino acid to form pyridoxamine phosphate and a keto acid. The amino group of pyri-doxamine phosphate can be transferred to another keto acid, regenerating pyridoxal phosphate. The vitamin is believed to play a role in the absorption of amino acids from the intestine. 

Most amino acids lose their nitrogen atom by a transamination reaction in which the -NH2 group of the amino acid changes places with the keto group of ct-ketoglutarate. The products are a new a-keto acid plus glutamate. The overall process occurs in two parts, is catalyzed by aminotransferase enzymes, and involves participation of the coenzyme pyridoxal phosphate (PLP), a derivative of pyridoxine (vitamin UJ. Different aminotransferases differ in their specificity for amino acids, but the mechanism remains the same. 

Pyridoxal phosphate mainly serves as coenzyme in the amino acid metabolism and is covalently bound to its enzyme via a Schiff base. In the enzymatic reaction, the amino group of the substrate and the aldehyde group of PLP form a Schiff base, too. The subsequent reactions can take place at the a-, (3-, or y-carbon of the respective substrate. Common types of reactions are decarboxylations (formation of biogenic amines), transaminations (transfer of the amino nitrogen of one amino acid to the keto analog of another amino acid), and eliminations. 

Figure 7-4. Ping-pong mechanism for transamination. E—CHO and E—CHjNHj represent the enzyme-pyridoxal phosphate and enzyme-pyridoxamine complexes, respectively. (Ala, alanine Pyr, pyruvate KG, a-ketoglutarate Glu, glutamate).

Pyridoxal phosphate is a coenzyme for many enzymes involved in amino acid metabolism, especially in transamination and decarboxylation. It is also the cofactor of glycogen phosphorylase, where the phosphate group is catalytically important. In addition, vitamin Bg is important in steroid hormone action where it removes the hormone-receptor complex from DNA binding, terminating the action of the hormones. In vitamin Bg deficiency, this results in increased sensitivity to the actions of low concentrations of estrogens, androgens, cortisol, and vitamin D. 

GOT (AST is the more recent abbreviation) catalyzes the transamination of 1-aspartic acid in the presence of a-ketoglut-aric acid, with pyridoxal phosphate being a required co-enzyme. The reaction is ... 

The Schiff base can undergo a variety of reactions in addition to transamination, shown in Fig. 6.4 for example, racemization of the amino acid via the a-deprotonated intermediate. Many of these reactions are catalyzed by metal ions and each has its equivalent nonmetallic enzyme reaction, each enzyme containing pyridoxal phosphate as a coenzyme. Many ideas of the mechanism of the action of these enzymes are based on the behavior of the model metal complexes. 

Glutamate can then participate in the formation of other amino acids via the process called transamination. Transamination is the exchange of the amino group from an amino acid to a keto acid, and provides the most common process for the introduction of nitrogen into amino acids, and for the removal of nitrogen from them. The reaction is catalysed by a transaminase enzyme, and the coenzyme pyridoxal phosphate (PLP) is required. 

Among the NH2 transfer reactions, transaminations (1) are particularly important. They are catalyzed by transaminases, and occur in both catabolic and anabolic amino acid metabolism. During transamination, the amino group of an amino acid (amino acid 1) is transferred to a 2-oxoacid (oxoacid 2). From the amino acid, this produces a 2-oxo-acid (a), while from the original oxoacid, an amino acid is formed (b). The NH2 group is temporarily taken over by enzyme-bound pyridoxal phosphate (PLP see p. 106), which thus becomes pyridoxamine phosphate. 

The active form of vitamin Be, pyridoxai phosphate, is the most important coenzyme in the amino acid metabolism (see p. 106). Almost all conversion reactions involving amino acids require pyridoxal phosphate, including transaminations, decarboxylations, dehydrogenations, etc. Glycogen phosphory-lase, the enzyme for glycogen degradation, also contains pyridoxal phosphate as a cofactor. Vitamin Be deficiency is rare. 

Vitamin Bg is a mixture of six interrelated forms pyridoxine (or pyridoxol) (Figure 19.23), pyri-doxal, pyridoxamine, and their 5 -phosphates derivatives. Interconversion is possible between all forms. The active form of the vitamin is pyridoxal phosphate, which is a coenzyme correlated with the function of more than 60 enzymes involved in transamination, deamination, decarboxylation, or desulfuration reactions. 

Pyridoxal phosphate is the coenzyme for the enzymic processes of transamination, racemization and decarboxylation of amino-acids, and for several other processes, such as the dehydration of serine and the synthesis of tryptophan that involve amino-acids (Braunstein, 1960). Pyridoxal itself is one of the three active forms of vitamin B6 (Rosenberg, 1945), and its biochemistry was established by 1939, in considerable part by the work of A. E. Braunstein and coworkers in Moscow (Braunstein and Kritzmann, 1947a,b,c Konikova et al 1947). Further, the requirement for the coenzyme by many of the enzymes of amino-acid metabolism had been confirmed by 1945. In addition, at that time, E. E. Snell demonstrated a model reaction (1) for transamination between pyridoxal [1] and glutamic acid, work which certainly carried with it the implication of mechanism (Snell, 1945). 

Isoniazid reacts with pyridoxal phosphate to form a hydrazone (Fig. 7.42), which is a very potent inhibitor of pyridoxal phosphate kinase. The hydrazone has a much greater affinity for the enzyme (100—lOOOx) than the normal substratepyridoxal. The result of this is a depletion of tissue pyridoxal phosphate. This cofactor is of importance particularly in nervous tissue for reactions involving decarboxylation and transamination. The decarboxylation reactions are principally affected however, with the result that transamination reactions assume a greater importance. 

FIGURE 18-4 Enzyme-catalyzed transaminations. In many aminotransferase reactions, a-ketoglutarate is the amino group acceptor. All aminotransferases have pyridoxal phosphate (PLP) as cofactor. Although the reaction is shown here in the direction of transfer of the amino group to a-ketoglutarate, it is readily reversible. 

The phosphate ester of the aldehyde form of vitamin B6, pyridoxal phosphate (pyridoxal-P or PLP), is required by many enzymes catalyzing reactions of amino acids and amines. The reactions are numerous, and pyridoxal phosphate is surely one of nature s most versatile catalysts. The story begins with biochemical transamination, a process of central importance in nitrogen metabolism. In 1937, Alexander Braunstein and Maria Kritzmann, in Moscow, described the transamination reaction by which amino groups can be transferred from one carbon skeleton to another.139 140 For example, the amino group of glutamate can be transferred to the carbon skeleton of oxaloacetate to form aspartate and 2-oxoglutarate (Eq. 14-24). 

Figure 14-5 Some reactions of Schiff bases of pyridoxal phosphate, (a) Formation of the quinonoid intermediate, (b) elimination of a (3 substituent, and (c) transamination. The quinonoid-carbanionic intermediate can react in four ways (1—4) if enzyme specificity and substrate structure allow.

There is an important biochemical counterpart of the deamination reaction that utilizes pyridoxal phosphate, 7, as the aldehyde. Each step in the sequence is catalyzed by a specific enzyme. The a-amino group of the amino acid combines with 7 and is converted to a keto acid. The resulting pyridoxamine then reacts to form an imine with a different a-keto acid, resulting in formation of a new a-amino acid and regenerating 7. The overall process is shown in Equation 25-6 and is called transamination. It is a key part of the process whereby amino acids are metabolized. 

The amino acid is then hydrolyzed to form an a-keto acid and pyridoxamine phosphate, the a-amino group having been temporarily transferred from the amino acid substrate on to pyridoxal phosphate (Fig. 5). These steps constitute one half of the overall transamination reaction. The second half occurs by a reversal of the above reactions with a second a-keto acid reacting with the pyridoxamine phosphate to yield a second amino acid and regenerate the enzyme-pyridoxal phosphate complex (Fig. 5). 

Other reactions that mimic the enzymic processes that require pyridoxal phosphate also have been realized. Werle and Koch reported the nonenzymic decarboxylation of histine (9). The racemization of alanine occurs in preference to its transamination when aqueous solutions with polyvalent cations are maintained at pH 9.5. Other amino acids are likewise racemized the order of rates is Phe, Met > Ala > Val > lieu. At lower pH, the dominant reaction is transamination, with pH maxima varying from 4.3-8 with the nature of the metal ion used as catalyst. 

Transamination, often also referred to as aminotransfer, is applied to those enzymatic reactions in which an amino group is exchanged between an amino acid and an a-keto acid. This type of reaction is catalyzed by a group of transferases called transaminases or aminotransferases. They are active in both the cytosol and the mitochondria of most cells. An essential prosthetic group of such enzymes is pyridoxal phosphate, and the reaction is generally of the ping-pong type. 

The ring nitrogen of pyridoxal phosphate exerts a strong electron withdrawing effect on the aldimine, and this leads to weakening of all three bonds about the a-carbon of the substrate. In nonenzymic reactions, all the possible pyridoxal-catalyzed reactions are observed - a-decarboxylation, aminotrans-fer, racemization and side-chain elimination, and replacement reactions. By contrast, enzymes show specificity for the reaction pathway followed which bond is cleaved will depend on the orientation of the Schiff base relative to reactive groups of the catalytic site. As discussed in Section 9.3.1.5, reaction specificity is not complete, and a number of decarboxylases also undergo transamination. 

To solve this problem, we used a mimic of a different enzyme, diaUcylglycine decarboxylase (30). In this enzyme, pyridoxal phosphate reacts with an alpha-disubstituted glycine to perform an irreversible decarboxylation (Fig. 6) while converting the pyridoxal species to a pyridoxamine. We imitated this with our model transaminations using pyridoxal species that carry hydrophobic chains, and we were able to achieve as many as 100 catalytic turnovers. Thus, we could imitate one enzyme—the ordinary transaminases—by also imitating another enzyme that solved the turnover problem. 

This reaction is crucial because it establishes the stereochemistry of the a-carbon atom (S absolute configuration) in glutamate. The enzyme binds the a-ketoglutarate substrate in such a way that hydride transferred from NAD(P)H is added to form the 1 isomer of glutamate (Figure 24,6). As we shall see, this stereochemistry is established for other amino acids by transamination reactions that rely on pyridoxal phosphate. 

The coenzyme form of pyridoxine is known as pyridoxal phosphate (PP) The most common type of reaction requiring PP as a coenz5mie is transamination. Enzymes catalysing such reactions are known as transaminases or aminotransferases. The coenzyme binds to its apoenzyme via Schiff s base between its aldehyde group and the epsilon amino group of a lysine in the... 

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