Pyridoxal phosphate-dependent reaction transamination

The experiments described earlier showed that in liver homogenates and extracts this reaction is brought about by transamination, which is an obligatory first step in the oxidation of tyrosine by such systems. The existence of su( h a transaminating system was already known (133, 134, 393), and the observed pyridoxal phosphate-dependence when transamination was was made rate-controlling (489) was in accordance with the known behavior of transaminases (c/. 482). 

Transamination Reactions of Other Pyridoxal Phosphate Enzymes In addition to their mean reactions, a number of pyridoxal phosphate-dependent enzymes also catalyze the half-reaction of tremstunination. Such enzymes include serine hydroxymethyltiemsfertise (Section 10.3.1.1), severed decarboxylases, and kynureninase (Section 8.3.3.2). 

These reactions are carried out by pyridoxal phosphate-dependent transami nases. Transamination reactions are required for the synthesis of most amino acids. 

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. 

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. 

The requirement for metal ions in these non-enzymatic transamination reactions suggests that such ions may be involved as catalysts in the enzymatic reactions. However, most of the pyridoxal phosphate dependent enzymes that have been purified to date do not contain metal ions, and addition of the latter to the reaction medium does not increase the rate of enzymatic reactions Metal ions, therefore, appear to fulfill some of the roles in non-enzymatic reactions played by the protein in enzymatic reactions, but the protein is apparently a much more efficient catalyst. 

The oxidative pathway of tryptophan metabolism is shown in Figure 3. Kynureninase is a pyridoxal phosphate-dependent enzyme, and in deficiency its activity is lower than that of tryptophan dioxygenase, so that there is an accumulation of hydroxy-kynurenine and kynurenine, resulting in greater metabolic flux through kynurenine transaminase and increased formation of kynurenic and xanthurenic acids. Kynureninase is exquisitely sensitive to vitamin Bg deficiency because it undergoes a slow inactivation as a result of catalysing the half-reaction of transamination instead of its normal reaction. The resultant enzyme with pyridoxamine phosphate at the catalytic site is catalytically inactive and can only be reactivated if there is an adequate concentration of pyridoxal phosphate to displace the pyridoxamine phosphate. 

E. coli (107, 125). The complexes have recently been reviewed (126). It is possible that lipoamide dehydrogenase also functions in the complexes that oxidatively decarboxylate the a-keto acids resulting from the transamination of valine, isoleucine, and leucine but these have proved difficult to resolve (127). Lipoamide dehydrogenase also functions in the pyridoxal phosphate and tetrahydrofolate-dependent oxidative decarboxylation of glycine in the anaerobic bacterium Peptococcus glyci-nophilus. The reaction in which the protein-bound lipoic acid is reduced is very complex and not yet fully understood the ultimate electron acceptor is NAD+ (112,113,128). 

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. 

The first step in the catabolism of most amino acids is the transfer of the o-amino group from the amino acid to a-ketoglutarate (tx-KG). This process is catalyzed by transaminase (aminotransferase) enzymes that require pyridoxal phosphate as a cofactor. The products of this reaction are glutamate (Glu) and the a-ketoacid analog of the amino acid destined for catabolic breakdown. For example, aspartate is converted to its a-keto analog, oxalo-acetate, by the action of aspartate transaminase (AST), which also produces Glu from a-KG. The transamination process is freely reversible, and the direction in which the reaction proceeds is dependent on the concentrations of the reactants and products. These reactions do not effect a net removal of amino nitrogen the amino group is only transferred from one amino acid to another. 

The pyridoxal phosphate (PLP)-dependent enzymes catalyze a diverse set of chemical transformations of amino acids. These include transamination, decarboxylation, and racemization reactions [Eqs. (41-43)],... 

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. 

The first step in tyrosine oxidation is a transamination to form p-hydroxyphenylpyruvic acid. Several groups of investigators independently showed a dependence of tyrosine oxidation on the presence of a keto acid. Knox and LeMay-Knox showed that a-ketoglutarate is a specific partner in the transamination and that pyridoxal phosphate is a cofactor in this reaction. Partial resolution of the transaminase allowed a demonstration of parallel restoration of transaminase activity and over-all tyrosine oxidation by addition of pyridoxal phosphate. 

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. 

Pyridoxal or its phosphate is known to catalyze as imine forms 57 a number of enzymatic transformations of a-amino acids (e.g., transamination). It is suggested that 1,3-dipolar species 58, tautomers of imines 57 (or their metal chelates), are involved in some pyridoxal-dependent enzymatic reactions (78TL2823). Thus, pyridoxal imines 57 react as N-unsubstituted azomethine ylide 1,3-dipoles 58 with iV-phenylmaleimide in boiling toluene or xylene to give the cycloadducts 59. 

The general arguments about the antiquity of cofactors apply to PLP. The nonenzymatic synthesis of pyridoxal under prebiotic conditions is considered possible, whereas the presence of a 5 phosphate group could hint to an ancestral attachment of the cofactor to RNA molecules. " Furthermore, there are specific grounds to assume that PLP arrived on the evolutionary scene before the emergence of proteins. In fact, in current metabolism, PLP-dependent enzymes play a central role in the synthesis and interconversion of amino acids, and thus they are closely related to protein biosynthesis. In an early phase of biotic evolution, free PLP could have played many of the roles now fulfilled by PLP-dependent enzymes, since the cofactor by itself can catalyze (albeit at a low rate) reactions such as amino acid transaminations, racemizations, decarboxylations, and eliminations. " This suggests that the appearance of PLP may have preceded (and somehow eased) the transition from primitive RNA-based life forms to more modern organisms dependent on proteins. 

A typical process flow diagram for the production of chiral amines using aminotransferases is shown in Figure 7.8. The buffering agent, pyridoxal 5-phosphate, amine donor, enzyme, and substrate ketone in aqueous solution are mixed in a biotransformation vessel. The desired reaction conditions such as temperature, pH, amine donor, and acceptor concentrations are maintained during transamination reaction. The transamination reaction time depends on the rate at which transaminase is catalyzing the reaction, while the extent of conversion depends on... 

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