Amino pyridoxal reaction

A modification of the pyridoxal—amino acid reaction (mentioned above) has been made for automatic analysis of amino acids by ligand-exchange chromatography [95]. This technique involves separation of the amino acids prior to fluorimetric reaction and determination. As the amino acids are eluted from the column, they are mixed with the pyridoxal-zinc(II) reagent to produce a highly fluorescent zinc chelate. Amounts of as low as 1 nmole of amino acid may be detected. The first reaction involved is the formation of the pyridoxyl-amino acid (Schiff base) as in Fig.4.46. The zinc then forms a chelate which probably has the structure shown in Fig. 4.48. 

Pyridoxal phosphate, the coenzyme of pyridoxine (vitamin B6) plays an important role in these reactions. Amino transferase reactions occur in two stages. 

Pyridoxal-5 phosphate (P-5 -P) and its amino analogue, pyridoxamine-5 -phosphate, function as coenzymes in the amino-transfer reactions. The P-5 -P is bound to the apoen-zyme and serves as a true prosthetic group. The P-5 -P bound to the apoenzyme accepts the amino group from the first substrate, aspartate or alanine, to form enzyme-bound pyridoxamine-5 -phosphate and the first reaction product, oxaloacetate or pyruvate, respectively. The coenzyme in amino form then transfers its amino group to the second substrate, 2-oxoglutarate, to form the second product, glutamate. P-5 -P is thus regenerated. 

Pyridoxal phosphate is the coenzyme in a large number of amino acid reactions. At this point it is convenient to consider together 1,he mechanism of those pyridoxal-dependent reactions concerned with aromatic amino acids. The reactions concerned are (1) keto acid formation (e.g., from kynurenine, above), 2) decarboxylation (e.g., of 5-hydroxytrypto-phan to 5-hydroxytryptamine, p. 106), (3) scission of the side claain (e.g., 3-tyrosinase, p. 78 tryptophanase, p. 110 and kynureninase, above), and 4) synthesis (e.g., of tryptophan from indole and serine, p. 40). Many workers have considered the mechanism of one or more of these reactions (e.g., 24, 216, 361, 595), but a unified theory is primarily due to Snell and his colleagues (summarized in 593). Snell s experiments have been carried out largely in vitro, and it should be emphasized that in vivo it is the enzyme protein which probably directs the electromeric changes. 

Amino acid metabolism requires the participation of three important cofactors. Pyridoxal phosphate is the quintessential coenzyme of amino acid metabolism (see Chapter 38). All amino acid reactions requiring pyridoxal phosphate occur with the amino group of the amino acid covalently bound to the aldehyde carbon of the coenzyme (Fig. 39.3). The pyridoxal phosphate then pulls electrons away from the bonds around the a-carbon. The result is transamination, deamination, decarboxylation, P-elimination, racemization, and -elimination, depending on which enzyme and amino acid are involved. 

Transaminases (also termed amino transferases [EC 2.6.l.X]) catalyze the redox-neutral amino-transfer reaction between an amine donor and a carbonyl group serving as acceptor (Scheme 2.225) [94, 1707-1712]. These enzymes require an activated benzaldehyde (pyridoxal-5 -phosphate, PLP, vitamin Bg) as cofactor, which functions as a molecular shuttle for the transfer of the NHa-moiety. In a first step, PLP forms a ketimine Schiff base with the amine-donor. Tautomerization of the C=N bond yields an aldimine, which is hydrolyzed to yield the cofactor in its aminated form (pyridoxamine, PMP). The latter reacts through the same order of events with the carbonyl group of the substrate to form the amine product and... 

Pyridoxal Derivatives. Various aldehydes of pyridoxal (Table 3) react with hemoglobin at sites that can be somewhat controlled by the state of oxygenation (36,59). It is thereby possible to achieve derivatives having a wide range of functional properties. The reaction, shown for PLP in Figure 3, involves first the formation of a Schiff s base between the amino groups of hemoglobin and the aldehyde(s) of the pyridoxal compound, followed by reduction of the Schiff s base with sodium borohydride, to yield a covalendy-linked pyridoxyl derivative in the form of a secondary amine. 

Bis-Pyndoxal Tetraphosphate. A second class of bifunctional reagents, described in 1988, involves two pyridoxal groups linked by phosphates of different lengths (89). As shown in Table 4, the yield of intramolecularly cross-linked hemoglobin increases dramatically with increasing length of the phosphate backbone. It is beheved that the site of reaction of (bis-PL) is between the amino-terminal amino group of one P-chain and the... 

An example of a biologically important aldehyde is pyridoxal phosphate, which is the active form of vitamin Bg and a coenzyme for many of the reactions of a-amino acids. In these reactions the amino acid binds to the coenzyme by reacting with it to form an imine of the kind shown in the equation. Reactions then take place at the amino acid portion of the imine, modifying the amino acid. In the last step, enzyme-catalyzed hydrolysis cleaves the imine to pyridoxal and the modified amino acid. 

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... 

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. 

The amino acid methionine is biosynthesized by a multistep roule that includes reaction of an inline of pyridoxal phosphate (PLP) to give an unsaturated imine. which then reacts with cysteine. What kinds of reactions are occurring in the two steps ... 

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. 

The metabolism of P-hydroxy-a-amino adds involves pyridoxal phosphate-dependent enzymes, dassified as serine hydroxymethyltransferase (SHMT) (EC 2.1.2.1) or threonine aldolases (ThrA L-threonine selective = EC 4.1.2.5, L-aHo-threonine selective = EC 4.1.2.6). Both enzymes catalyze reversible aldol-type deavage reactions yielding glycine (120) and an aldehyde (Eigure 10.45) [192]. 

Pantothenic acid is present in coenzyme A and acyl carrier protein, which act as carriers for acyl groups in metabolic reactions. Pyridoxine, as pyridoxal phosphate, is the coenzyme for several enzymes of amino acid metabolism, including the aminotransferases, and of glycogen phosphorylase. Biotin is the coenzyme for several carboxylase enzymes. 

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 role of Schiff bases formed between pyridoxal phosphate and amino acid residues as intermediate products in many enzymatic reactions is well known and documented. NMR is an excellent tool for studies of the enzymatic processes involving Schiff bases formation. 

These enzymes invariably involve a cofactor, pyridoxal phosphate (vitamin B6). In addition, pyridoxal phosphate is also required for most decarboxylations, racemizations, or elimination reactions in which an amino acid is a substrate. Pyridoxal phosphate is not involved in decarboxylations in which the substrate is not an amino acid. So if a question... 

In addition to a-allenic a-amino acids, the corresponding allenic derivatives of y-aminobutyric acid (GABA) have also been synthesized as potential inhibitors of the pyridoxal phosphate-dependent enzyme GABA-aminotransferase (Scheme 18.49) [131,138-142]. The synthesis of y-allenyl-GABA (152) and its methylated derivatives was accomplished through Crabbe reaction [131], aza-Cope rearrangement [138] and lactam allenylation [139], whereas the fluoroallene 153 was prepared by SN2 -reduc-tion of a propargylic chloride [141]. 

Identification of pyridoxal phosphate as coenzyme suggested the aldehyde group on pyridoxine might form an intermediate Schiff s base with the donor amino acid. Pyridoxamine phosphate thus formed would in turn donate its NH2 group to the accepting a-ketonic acid, a scheme proposed by Schlenk and Fisher. 15N-labeling experiments and, later, the detection of the Schiff s base by its absorption in UV, confirmed the overall mechanism. Free pyridoxamine phosphate however does not participate in the reaction as originally proposed. Pyridoxal phosphate is invariably the coenzyme form of pyridoxine. 

---