Pyridoxal phosphate in transamination

What is the role of pyridoxal phosphate in transamination reactions ... 

Thus the mechanism of action of pyridoxal phosphate in transamination must be left open. There is as yet no clue to its mechanism of action in decarboxylation and in the tryptophanase reaction. 

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. 

It is involved as a coenzyme (pyridoxal phosphate) in metabolism of tryptophan, in several metabolic transformations of amino acids including transamination, decarboxylation and racemization. 

The required coenzymes are pyridoxal phosphate in the transamination reaction and NAD+/NADH in the redox reactions. 

Indolmydn.—Previous evidence on the biosynthesis of indolmycin (88) in Strepto-myces griseus cultures accords with the pathway shown in Scheme 4. The first two steps in the pathway have been carried out using cell-free extracts of 5. griseus - and recent work has led to the isolation of two enzymes which can effect these transformations. The first, tryptophan transaminase, catalysed the pyridoxal phosphate-dependent transamination of L-tryptophan, but not D-trptophan, and in common with some other microbial transaminases, a-ketoglutarate was an efficient amino-group acceptor. L-Phenylalanine, tyrosine, and 3-methyltryptophan (this compound inhibited enzyme function) also underwent transamination. 

The same scaffold was used to design catalysts for pyridoxal phosphate-dependent deamination of aspartic acid to form oxaloacetate, one half of the transamination reaction [8], and oxaloacetate decarboxylation [14]. Catalysis was due to binding of pyridoxal phosphate in close proximity to His residues capable of rate limiting 1,3 proton transfer. A two-residue catalytic site containing one Arg and one Lys residue was found to be the most efficient decarboxylation agent, more efficient per residue than the Benner catalyst, most likely due to a combination of efficient imine formation, pK depression and transition state stabilization. 

During transamination reactions, the a-amino group is transferred to the coenzyme pyridoxal phosphate. In the last part of the reaction, the a-amino group is transferred from pyridoxal phosphate to an a-keto acid. 

Snell s observations of the formation of pyridoxamine by heating vwth a-amino acids suggested that pyridoxal is involved in transamination reactions. As an enzyme cofactor it could transfer an a-amino group from an amino acid to the a-keto group of an a-keto acid. It is now established that the action of pyridoxal phosphate in aminotransferase enzymes includes formation of pyridoxamine phosphate during the catalytic cycle (see text, p. 641)... 

Fig. 17.14 Participation of the cofactor pyridoxal phosphate in the process of transamination.

At pH values where the enzyme is bound to pyridoxal as a SchifF base (40), reaction with sodium borohydride destroys the activity of the enzyme by reducing the SchifF base to a pyridoxylamine (42). This observation gives strength to the hypothesis that the subsequent reaction of enzyme with an amino acid involves transamination. Skeletal muscle phosphorylase is an exception , indicating that this particular enzyme does not require a SchifF base linkage for its activity. Further research is required to determine whether the role of pyridoxal phosphate in this enzyme can be attributed simply to maintenance of the active site of the enzyme in the required conformation or whether a new set of catalytic properties must be ascribed to the vitamin Bg aldehyde. 

Fio. 1. Role of pyridoxal phosphate and pyridoxamine phosphate in transamination. 

Fiq. 1. Proposed mechanism for the role of pyridoxal phosphate and pyridoxa-mine phosphate in transamination (SH). 

Although it is superficially attractive to write a mechanism that consists of formation of an imine, tautomerization, and hydrolysis, this is not what happens nor does it explain the need for pyridoxal phosphate in the reaction. The pyridoxal phosphate is bonded to a lysine of the enzyme as an imine (14.25). In the transamination reaction, another free amino acid displaces the lysine that is part of the enzyme from pyridoxal, in an imine exchange reaction (Figure 14.33). 

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. 

The mechanism of the first part of transamination is shown in Figure 29.14. The process begins with reaction between the a-amino acid and pyridoxal phosphate, which is covalently bonded to the aminotransferase by an iminc linkage between the side-chain -NTI2 group of a lysine residue and the PLP aldehyde group. Deprotonation/reprotonation of the PLP-amino acid imine in steps 2 and 3 effects tautomerization of the imine C=N bond, and hydrolysis of the tautomerized imine in step 4 gives an -keto acid plus pyridoxamine... 

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. 

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. 

We have just noted the role that pyridoxal phosphate plays as a coenzyme (cofactor) in transamination reactions (see section 15.6). Pyridoxal 5 -phosphate (PLP) is crucial to a number of biochemical reactions. PLP, together with a number of closely related materials that are readily converted into PLP, e.g. pyridoxal, pyridoxine and pyridoxamine, are collectively known as vitamin Bg, which is essential for good health. 

Pyridoxal phosphate (4) is the most important coenzyme in amino acid metabolism. Its role in transamination reactions is discussed in detail on p. 178. Pyridoxal phosphate is also involved in other reactions involving amino acids, such as decarboxylations and dehydrations. The aldehyde form of pyridoxal phosphate shown here (left) is not generally found in free form. In the absence of substrates, the aldehyde group is covalently bound to the e-amino group of a lysine residue as aldimine ( Schiffs base ). Pyridoxamine phosphate (right) is an intermediate of transamination reactions. It reverts to the aldehyde form by reacting with 2-oxoacids (see p. 178). 

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. 

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. 

An early step in the catabolism of amino acids is the separation of the amino group from the carbon skeleton. In most cases, the amino group is transferred to a-ketoglutarate to form glutamate. This transamination reaction requires the coenzyme pyridoxal phosphate. 

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