Pyridoxal transamination

Finally, a pyridoxal transamination converts the two keto-acids stereospecifically to the corresponding amino acids, valine (R = Me) and isoleucine (R = Et). The donor amino acid is probably glutamate—-it usually is in amino acid synthesis. 

The biologically active form of vitamin Bg is pyridoxal-5-phosphate (PEP), a coenzyme that exists under physiological conditions in two tautomeric forms (Figure 18.25). PLP participates in the catalysis of a wide variety of reactions involving amino acids, including transaminations, a- and /3-decarboxylations, /3- and ") eliminations, racemizations, and aldol reactions (Figure 18.26). Note that these reactions include cleavage of any of the bonds to the amino acid alpha carbon, as well as several bonds in the side chain. The remarkably versatile chemistry of PLP is due to its ability to... 

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. 

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).

Be Pyridoxine, pyridoxal, pyridoxamine Coenzyme in transamination and decarboxylation of amino acids and glycogen phosphorylase role in steroid hormone action Disorders of amino acid metabolism, convulsions... 

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 ... 

Another interesting example is SHMT. This enzyme catalyzes decarboxylation of a-amino-a-methylmalonate with the aid of pyridoxal-5 -phosphate (PLP). This is an unique enzyme in that it promotes various types of reactions of a-amino acids. It promotes aldol/retro-aldol type reactions and transamination reaction in addition to decarboxylation reaction. Although the types of apparent reactions are different, the common point of these reactions is the formation of a complex with PLP. In addition, the initial step of each reaction is the decomposition of the Schiff base formed between the substrate and pyridoxal coenzyme (Fig. 7-3). 

The deamination of primary amines such as phenylethylamine by Escherichia coli (Cooper et al. 1992) and Klebsiella oxytoca (Flacisalihoglu et al. 1997) is carried out by an oxidase. This contains copper and topaquinone (TPQ), which is produced from tyrosine by dioxygenation. TPQ is reduced to an aminoquinol that in the form of a Cu(l) radical reacts with O2 to form H2O2, Cu(ll), and the imine. The mechanism has been elucidated (Wihnot et al. 1999), and involves formation of a Schiff base followed by hydrolysis in reactions that are formally analogous to those involved in pyridoxal-mediated transamination. 

Vitamin Ba (pyridoxine, pyridoxal, pyridoxamine) like nicotinic acid is a pyridine derivative. Its phosphorylated form is the coenzyme in enzymes that decarboxylate amino acids, e.g., tyrosine, arginine, glycine, glutamic acid, and dihydroxyphenylalanine. Vitamin B participates as coenzyme in various transaminations. It also functions in the conversion of tryptophan to nicotinic acid and amide. It is generally concerned with protein metabolism, e.g., the vitamin B8 requirement is increased in rats during increased protein intake. Vitamin B6 is also involved in the formation of unsaturated fatty acids. 

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. 

The intervention of a metal ion in the stoichiometry of a reaction has been illustrated several times previously. Reaction is forced to completion in ester hydrolysis since the carboxylate grouping forms a more stable complex than the ester moiety does. A similar driving force underlies the formation of macrocycles and the completion of transamination by formation of the metal-Schiff base complex. The latter is particularly relevant in dilute solution and at low pH. For example, the extent of aldimine formation between pyridoxal and alanine is undetectable at the physiological pH but occurs to the extent of = 10% in the presence of zinc... 

Mechanistically, transamination by the free coenzyme proceeds through a number of discrete steps as illustrated in Fig. 3. The first step of the process, aldi-mine formation (Fig. 3, Step I), is common to all pyridoxal-dependent reactions. The rate and extent of this reaction are influenced by factors including reactant concentrations, the nature of the amino acid, pH, and solvent. However, it is important to realize that the coenzyme itself facilitates Schiff base formation in... 

Fig. 2. Catalytic cycle of pyridoxal/pyridoxamine-dependent transamination...

This designed construct, as such, may not influence subsequent steps of transamination due to loss of the strong association between the coenzyme and the synthetic peptide after amino acid binding. However, the selectivity of the peptide for pyridoxal phosphate reveals the potential power of peptide design and the importance of secondary binding interactions for defining the formation of specific binary complexes. 

Work in the Imperiali laboratory has also focused on exploring the ability of minimal peptide scaffolds to augment the rate of coenzyme-mediated transaminations [22-25]. To accomplish this, a strategy has been developed in which the core functionality of the coenzyme is incorporated as an integral constituent of an unnatural coenzyme amino acid chimera construct. Thus, non-cova-lent binding of the coenzyme to the peptide or protein scaffold is unnecessary. Both the pyridoxal and pyridoxamine analogs have been synthesized in a form competent for Fmoc-based solid phase peptide synthesis (SPPS) (Fig. 7) [23,24]. 

Table 2. Observed rate constants for the transamination of pyruvate to alanine mediated by various pyridoxal derivatives...

The terminology vitamin Bg covers a number of structurally related compounds, including pyridoxal and pyridoxamine and their 5 -phosphates. Pyridoxal 5 -phosphate (PLP), in particular, acts as a coenzyme for a large number of important enzymic reactions, especially those involved in amino acid metabolism. We shall meet some of these in more detail later, e.g. transamination (see Section 15.6) and amino acid decarboxylation (see Section 15.7), but it is worth noting at this point that the biological role of PLP is absolutely dependent upon imine formation and hydrolysis. Vitamin Bg deficiency may lead to anaemia, weakness, eye, mouth, and nose lesions, and neurological changes. 

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. 

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. 

This pyridoxal 5-phosphate-dependent enzyme [EC 2.6.1.4] catalyzes the transamination of glyoxylate from L-glutamate to produce glycine and a-ketoglutarate. 

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. 

GABA synthesis inhibitors act on the enzymes involved in the decarboxylation and transamination of GABA. Glutamic acid decarboxylase (GAD), the first enzyme in GABA biosynthesis, is inhibited easily by carbonyl reagents such as hydrazines [e.g., hydrazinopropionic acid (4.164) or isonicotinic acid hydrazide (4.165)], which trap pyridoxal, the essential cofactor of the enzyme. A more specific inhibitor is allylglycine (4.166). All of these compounds cause seizures and convulsions because they decrease the concentration of GABA. 

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

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). 

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