Pyridoxal phosphate-dependent reaction elimination

This pyridoxal-phosphate-dependent enzyme [EC 4.2.99.9], also known as cystathionine y-synthase, catalyzes the reaction of O-succinyl-L-homoserine with L-cysteine to produce cystathionine and succinate. The enzyme can also use hydrogen sulfide and methanethiol as substrates, producing homocysteine and methionine, respectively. In the absence of a thiol, the enzyme can also catalyze a /3,y-elimination reaction to form 2-oxobu-tanoate, succinate, and ammonia. 

Due to the absence of a hydrogen atom on the a-carbon, the a-fluoroalkyl amino acids (except, of course, the fluoroalanines, vide supra) cannot undergo an elimination of HR Consequently, they are more stable than fluoroalanines and other jS-fluoro amino acids previously described. On the other hand, similar to proteogenic amino acids, jS-fluoro amino acids and a-fluoroalkyl amino acids are generally substrates of pyridoxal phosphate depending on enzymes such as racemases and decarboxylases. When an amino acid is a substrate of such enzymes, the enzyme induces the development of a negative charge on the a-carbon, which can initiate a /(-elimination process. This reaction affords an electrophilic species (Michael acceptor type), which is able to add a nucleophilic residue of the enzyme. This notion of mechanism-based inhibitor is detailed in Chapter 7. 

The glycolytic pathway includes three such reactions glucose 6-phosphate isomer-ase (1,2-proton transfer), triose phosphate isomerase (1,2-proton transfer), and eno-lase (yS-elimination/dehydration). The tricarboxylic acid cycle includes four citrate synthase (Claisen condensation), aconitase (j5-elimination/dehydration followed by yS-addition/hydration), succinate dehydrogenase (hydride transfer initiated by a-proton abstraction), and fumarase (j5-elimination/dehydration). Many more reactions are found in diverse catabolic and anabolic pathways. Some enzyme-catalyzed proton abstraction reactions are facilitated by organic cofactors, e.g., pyridoxal phosphate-dependent enzymes such as amino acid racemases and transaminases and flavin cofactor-dependent enzymes such as acyl-C-A dehydrogenases others. 

Pyridoxal 5 -phosphate-dependent a, -elimination reactions of O-acetylserine sulfhydrylase 01ACR49. 

Ibere is no doubt that such a reaction, catalysed by a pyridoxal phosphate-dependent enzyme, can occur in biological systems. It is quite possible, however, that this only represents a side reaction of other enzymes. Qrstathionase, for example, will act on cystine with the elimination of a cysteine persulphide and pyruvate. The persulphide then reacts with cysteine to eliminate sulphide and re nerate cystine. The complete cycle would constitute a cysteine desulphydrase activity. 

Applications of tryptophan synthetase Tryptophan synthetase (EC 4.2.1.20) is a pyridoxal phosphate-dependent enzyme that, in the cell, catalyzes the a,/3-elimination of water from serine to form a pyridoxyl-bound a-aminoacrylate, which undergoes Michael addition of indole to form the named amino acid. This type of reaction has been used to prepare (5)-tryptophan isotopomers with a variety of labeling patterns by use of different labeled indoles and (5)-serines in yields of up to 98% based on indole and 92% based on (5)-serine. 

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. 

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. 

Condensation of L-alanine with a heptaketide CoA thioester and concomitant decarboxylation gave compound 31, which was formed with overall retention of the absolute confguration. The same behavior was verified in reactions catalyzed by enzymes of the a-oxoamine synthetase family which depend on pyridoxal phosphate (PLP) as the cofactor. Ring closure, reduction of carbonyl at position 3, and subsequent elimination of water lead to 2//-azepine 33. The latest steps of the biosynthesis involve the modification of the side chain, leading to the carbonyl group of 30 [21],... 

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. 

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