Pyridoxal phosphate, with amino acids

NMR studies have been carried out on Schiff bases derived from pyridoxal phosphate and amino acids, since they have been proposed as intermediates in many important biological reactions such as transamination, decarboxylation, etc.90 The pK.d values of a series of Schiff bases derived from pyridoxal phosphate and a-amino adds, most of which are fluorinated (Figure 11), have been derived from H and19F titration curves.91 The imine N atom was found to be more basic and more sensitive to the electron-withdrawing effect of fluorine than the pyridine N atom. Pyridoxal and its phosphate derivative are shown in Figure 12a. The Schiff base formation by condensation of both with octopamine (Figure 12b) in water or methanol solution was studied by 13C NMR. The enolimine form is favoured in methanol, while the ketoamine form predominates in water.92... 

Transaminase enzymes react pyridoxamine phosphate with ketoacids to form pyridoxal phosphate and amino acids (Figure 2.9). This is the first step in an overall transamination... 

FIGURE 18.27 Pyridoxal-5-phosphate forms stable Schiff base adducts with amino acids and acts as an effective electron sink to stabilize a variety of reaction intermediates. 

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]. 

The principles of the above reactions form the basis of a series of important metabolic interconversions involving the coenzyme pyridoxal phosphate (structure 2.41). This condenses with amino acids to form a Schiff base (structure 2.42). The pyridine ring in the Schiff base acts as an electron sink which very effectively stabilizes a negative charge. 

As discussed in Chapter 2, section C2, pyridoxal phosphate condenses with amino acids to form a Schiff base (structure 8.44). Each of the three groups around the chiral carbon at the top of structure 8.44 may be cleaved to give an anion that is stabilized by delocalization of the electrons over the 7r orbitals. 

Figure 9.2. Reactions of pyridoxal phosphate-dependent enzymes with amino acids.

Mechanism of the first phase of transamination. The -NH2 group from the amino acid is transferred to pyridoxal phosphate, with formation of the corresponding a-keto acid. The second phase occurs by the reversal of the first phase reactions and is initiated by formation of a Schiff base with the a-keto acid substrate and pyridoxamine phosphate. The transamination cycie is compieted with formation of the corresponding a-amino acid and pyridoxal phosphate. 

The reaction mechanism consists of formation of a Schiff base by pyridoxal phosphate with a reactive amino group of the enzyme entry of glycine and formation of an enzyme-pyridoxal phosphate-glycine-Schiff base complex loss of a proton from the a carbon of glycine with the generation of a carbanion condensation of the carbanion with succinyl-CoA to yield an enzyme-bound intermediate (a-amino-yS-ketoadipic acid) decarboxylation of this intermediate to ALA and liberation of the bound ALA by hydrolysis. ALA synthesis does not occur in mature erythrocytes. 

Perhaps the best characterized organic cofactor-dependent racemase is alanine racemase, which employs pyridoxal 5 -phosphate (PLP) (Table 7.1). o-alanine is necessary for the synthesis of the peptidoglycan layer of bacterial cell walls in Gram negative and positive bacteria [1]. Alanine racemase is thus a ubiquitous enzyme in bacteria and an excellent drug target [2]. Both its crystal structure and mechanism have been well investigated. PLP reacts with amino acids to produce... 

The vitamin is transformed into pyridoxal phosphate (PLP), which, as a pyridine, is basic at the ring nitrogen, and in the active form, is V-protonated. Enzymes containing PLP have various functions, all connected with amino acids. Amongst other activities, PLP-containing enzymes can (i) effect transfer of an amino group from an a-amino acid to an a-keto acid (ii) bring about decarboxylation of an a-amino acid or... 

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. 

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... 

Pyridoxal phosphate (PLP) is the reactive center of several enzymes. Its reactive group is the formyl group, which is positioned para to a pyridine nitrogen. Schiff bases with amino acids are stabilized by the 3-hydroxy group, and two quinonoid structures may occur. Pyridoxal absorbs at 320 nm (E = 4000), and the... 

Pyridoxal phosphate bound to enzyme in Schiff-base linkage with amino acid... 

Schiff base formation between pyridoxal (140) and amino acids leads to complexes of type (141) which are in tautomeric equilibrium with (142). This tautomeric equilibrium leads to transamination, thus the same metal complexes can be obtained when either pyridoxal and alanine or pyridoxamine and pyruvic acid are allowed to react together in the presence of a metal ion. Hopgood" has studied the rates of transamination of 15 amino acids in the presence of zinc(II) and pyridoxal 5-phosphate (143). On mixing the reagents zinc(II)-aldimine complexes are rapidly formed (ca. 5 min) and these species subsequently transaminate in a slow second step. Ai" and Zn" systems have been particularly well studied.The role of the metal ion seems to involve both stabilization or trapping of the Schiff base, and in addition it also ensures the planarity of the conjugated ir-system. In the case of the aldimine tautomer, extensive H NMR studies have shown that formation of the ternary complex results in activation at the amino acid 2-carbon. At room temperature the reaction occurs without incorporation of into the aldehyde methine position indicating that the primary mechanism is carbanion formation rather than tautomerism. 

Some coenzymes participate in group-transfer reactions that are difficult to carry out with amino acid side chain chemistries alone. For example, none of the side chains of the normal 20 amino acids can accept an amino group easily. On the other hand, the coenzyme pyridoxal phosphate has a carbonyl group that is well adapted to accepting or donating amino groups. 

Early work by Esmond Snell on the enzymes that employ pyridoxal phosphate included experiments in which free pyridoxal was heated with amino acids like glutamate. Snell found that the (-amino group of glutamate was transferred to pyridoxal, generating pyridoxamine. Why was this an important clue to the function of pyridoxal as a cofactor ... 

It is obvious that AOA can be considered the aminooxy analogue of glycine, while AOPP is the aminooxy analogue of phenylalanine. Both of these compounds can be expected to interfer with amino acid metabolizing enzymes carrying a carbonyl group(e.g. that of pyridoxal phosphate in the case of transaminases or dehydroalanine in... 

Because of the numerous enzymes requiring pyridoxal phosphate, a large variety of biochemical lesions are associated with vitamin B5 deficiency. These lesions are concerned primarily with amino acid metabolism, and a deficiency affects the animal s growth rate. Convulsions may also occur, possibly because a reduction in the activity of glutamic acid decarboxylase results in an accumulation of glutamic acid. In addition, pigs reduce their food intake and may develop anaemia. Chicks on a deficient diet show jerky movements in adult birds, hatchability and egg production are adversely affected. In practice, vitamin B5 deficiency is unlikely to occur in farm animals because of the vitamin s wide distribution. 

Preexistence of a Schiff base between pyridoxal phosphate and the enzyme may account for the greatly enhanced rates of enzymatic reactions is compared to the rates of the corresponding non-enzymatic reactions. Subsequent reactions of the enzyme with amino acids must involve Schiff base formation via a fast transimination step (see section IV.D). Once the new Schiff base is formed, the e-amino group of the lysine residue that was originally bound to pyridoxal phosphate is free and is in a favorable position to act as a catalyst in subsequent steps of the enzymatic reaction. 

Vitamin Bg occurs in the three forms shown below. In the form of pyridoxal phosphate the vitamin acts as a coenzyme for more than sixty enzymes concerned with amino acid metabolism, e.g. aminotransferases, decarboxylases, deaminases and desulphurases. It also plays a role in the absorption of amino acids and is a constituent of glycogen phosphorylase. 

A role for a-keto-c-aminopimelic acid on the pathway of lysine syntheds is indicated by the discovery of a transaminase that acts on both diaminopimelic acid and on lysine 194). Cell suspendons of acetone-dried bacteria at pH 5 were able to transaminate all three stereoisomers of diaminopimelic acid and also d- and L-lysine in the presence of pyridoxal phosphate, with pyruvate, oxalacetate, and a-ketoglutarate serving as amino group acceptors. 

Imino acids are easily decarboxylated (188) and the amine could then be set free either through hydrolysis or through displacement by a second molecule of amino acid. None of the amino acid decarboxylases has been prepared in pure form, but Werle and Heitzer (186) have purified histidine decarboxylase about 35-fold. Recently, pyridoxal phosphate was shown to be the prosthetic group of several amino acid decarboxylases (7,74,162). For optimal yields on amines, incubations with amino acid decarboxylases were carried out under anaerobic conditions (91) in order to prevent the action of amine oxidase, and, in rat kidneys, also that of Z-amino acid oxidase. 

Pyridoxal 5 -phosphate (PLP, Figure 14.32) is a cofactor of enzymes that are responsible for amino acids transformations such as racemization, transamination, and decarboxylation. Its enzymology has been reviewed recently [86, 87]. When PLP is embedded in an enzyme it usually forms the so-called internal aldimine with the e-group of a lysine residue in the active site, or an external aldimine with amino acid substrates or inhibitors. The similarity with the Schiff... 

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