Pyridoxal 5 -phosphate racemization

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... 

Novozymes, a subtilisin produced by Bacillus licheniformis, was used by Chen et al ° to carry out a dynamic kinetic resolution of benzyl, butyl, or propyl esters of DL-phenylalanine, tyrosine, and leucine. The hydrolysis was performed at pH 8.5 in 2-methyl-2-propanol/water (19 1) and the freed L-amino acids precipitated. The key feature bringing about continual racemization of the remaining D-amino acid esters was the inclusion of 20 mmol 1 pyridoxal phosphate. 

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. 

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). 

T Although D-amino acids do not generally occur in proteins, they do serve some special functions in the structure of bacterial cell walls and peptide antibiotics. Bacterial peptidoglycans (see Fig. 20-23) contain both D-alanine and D-glutamate. D-Amino acids arise directly from the l isomers by the action of amino acid racemases, which have pyridoxal phosphate as cofactor (see Fig. 18-6). Amino acid racemization is uniquely important to bacterial metabolism, and enzymes such as... 

Bacteria synthesize D-amino acids from L-amino acids in racemization reactions requiring pyridoxal phosphate. 

It is well over 40 years since Pfeiffer discovered that certain reactions of a-amino acid esters, in particular, ester exchange, racemization and oxygenation, are effected very readily when their Schiff bases with salicylaldehyde are complexed to a transition metal ion (most notably Cu11). The Schiff bases result from a condensation reaction between a reactive carbonyl group and the amino group of the amino acids. Snell and his co-workers43 were also one of the first to point out that similar reactions also occurred if pyridoxal was used instead of salicylaldehyde, and that there is a close analogy with pyridoxal phosphate-promoted enzymic reactions of a-amino acid metabolism. Since then much work has been due on these and other similar systems and their reactivities. 

The reactions catalyzed by transaminases are anergonic as they do not require an input of metabolic energy. They are also freely reversible, the direction of the reaction being determined by the relative concentrations of the amino acid-keto acid pairs. Pyridoxal phosphate is not just used as the coenzyme in transamination reactions, but is also the coenzyme for several other reactions involving amino acids including decarboxylations, deaminations, racemizations and aldol cleavages. 

A number of decarboxylase enzymes have been described as catalysts for the preparation of chiral synthons, which are difficult to access chemically (see Chapter 2).264 The amino acid decarboxylases catalyze the pyridoxal phosphate (PLP)-dependent removal of C02 from their respective substrates. This reaction has found great industrial utility with one specific enzyme in particular, L-aspartate-P-decarboxylase (E.C. 4.1.1.12) from Pseudomonas dacunhae. This biocatalyst, most often used in immobilized whole cells, has been utilized by Tanabe to synthesize L-alanine on an industrial scale (multi-tons) since the mid-1960s (Scheme 19.33).242-265 Another use for this biocatalyst has been the resolution of racemic aspartic acid to produce L-alanine and D-aspartic acid (Scheme 19.34). The cloning of the L-aspartate-P-decarboxylase from Alcaligenes faecalis into E. coli offers additional potential to produce both of these amino acids.266... 

Other reactions that mimic the enzymic processes that require pyridoxal phosphate also have been realized. Werle and Koch reported the nonenzymic decarboxylation of histine (9). The racemization of alanine occurs in preference to its transamination when aqueous solutions with polyvalent cations are maintained at pH 9.5. Other amino acids are likewise racemized the order of rates is Phe, Met > Ala > Val > lieu. At lower pH, the dominant reaction is transamination, with pH maxima varying from 4.3-8 with the nature of the metal ion used as catalyst. 

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. 

Like modular PKSs, peptide synthetases also epimerize some substrates and/or intermediates. For example, the starter substrate amino acid of cyclosporin A is D-Ala. Racemization of alanine is not catalyzed by an integrated subunit of cyclosporin A synthetase, but by alanine racemase. This is a separate, pyridoxal phosphate-dependent enzyme [ 193]. In contrast, Grsl and Tycl covalently activate L-Phe as a thioester and subsequently epimerize the amino acid [194]. D-Phe is the only epimer accepted as a substrate for dipeptide formation by Grs2 and Tyc2 [195, 196]. No racemization activity is detected in a pantetheine-deficient mutant of Grsl [197]. Deletion mutagenesis pointed to the requirement of the COOH-terminal part of the module for epimerizing L-Phe to D-Phe [180]. In contrast, the biosynthesis of actinomycin D, a bicyclic chromo-pentapeptide lactone (Fig. 10), involves formation of the dipeptide 6-MHA (methylanthranilic acid)-L-Thr-L-Val prior to epimerization of the L-Val exten-... 

The late 1990s saw the development of an alternative methodology for the enzymatic resolution of racemic amines using transaminases. Transaminases are pyridoxal phosphate 50 dependent enzymes that catalyze the transfer of an amine group to a carbonyl compound (amine group acceptor), such as a ketone, aldehyde, or keto add (Figure 14.19). 

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)],... 

The most versatile of the coenzymes is perhaps pyridoxal phosphate (PEP). The PEP containing enzymes catalyze a wide variety of reactions such as racemization, transamination, [3- and a-decarboxylation, and interconversion of side chains. The first step of all these reactions is the transition between an internal aldimine intermediate to an external aldimine intermediate, which involves the condensation of PEP with an external amino acid substrate to form a Schiff base. The internal aldimine intermediate can then either undergo a-decarboxylation to convert the amino acid substrate into amines and aldehydes, or lose the a-hydrogen... 

Pyridoxal phosphate participates in transaminations, decarboxylations, racemizations, and numerous... 

Pyridoxine (vitamin B5) Pyridoxal phosphate (PLP) Dec arboxylation Transamination Racemization —C bond cleavage a,B-Elimination -Substitution Anemia... 

We have seen that amino acid racemization is catalyzed by an enzyme that requires pyridoxal phosphate as a coenzyme (Section 25.6). What we need, then, is a compound that will inhibit this enzyme. Because the natural substrate for the enzyme is an amino acid, an amino acid analog should be a good inhibitor. 

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. 

Figure 11.13 Reactions at a-carbon of a-amino acids catalyzed by pyridoxal enzymes All three substituents at C are subject to labilization in the three types of a-carbon reactions. The hydrogen is labilized in recemization reactions, the amino group is labUized in the transamination and the carboxyl group is labilized in decarboxylation. a-Amino acid condenses with pyridoxal phosphate to yield pyridoxylidene imino acid (an aldimine). The common intermediate, aldimine and distinct ketimines leading to the production of oxo-acid (in transamination), amino acid (in racemization) and amine (in decarboxylation) are shown. The catalytic acid (H-A-) and base (-B ) are symbolic both can be from the same residue such as Lys258 in aspartate aminotransferase.

Like homogeneous catalysis, the removal of a-hydrogen of the amino acid fragment by OH ions, the local concentration of which is apparently high in the polymer phase, is probably the rate-determining step of heterogeneous racemization. Under similar conditions, the rate of a-amino acid racemization decreases in the sequence Ala = Ser>Phe>Nva>Lys>Val, and correlates with the rate of substrate racemization in the presence of Schiff bases and transamination of amino acids by pyridoxal phosphate. 

Schiff base formation between pyridoxal phosphate and amino acids are the basis for most enzymatic transformations of amino acids including transamination, decarboxylation, and racemization. Schiff bases formed between amino acids and pyridoxal phosphate or other heteroaromatic or aromatic aldehydes are, however, not only transformed enzymatically, but can, without enzymatic catalysis, undergo a large number of reactions, although at lower rate and/or higher temperatures than those for the corresponding enzymatic reactions. The enzymatic reactions require metal ions as cofactors and in analogy the nonenzymatic reaction are also catalyzed by metal ions, most effectively by cupric ions. 

Pyridoxine Nicotinic acid Pyridoxal phosphate Coenzyme for transamination, decarboxylation and racemization... 

Pyridoxal phosphate is a necessary coenzyme for a number of different biochemical reactions transamination, amino acid oxidation, amino acid decarboxylation, glycogen breakdown, and racemization of d- and L-amino acids. 

In the preceding sections pyridoxal phosphate has appeared as an essential cofactor for many apparently unrelated processes. It has been possible, however, to demonstrate common features to many different reactions of amino acids decarboxylation, transamination, racemization, substitution, and elimination. The properties of pyridoxal are such that many of the enzymatic reactions have been duplicated nonenzymatically... 

Racemizations. When pyridoxal forms a Schiff s base with an amino acid in the presence of a metal ion, the structure of (II) seems likely to occur. Ionization of the a-hydrogen is favored by the formation of the quinoid structure on the right. In the racemization of amino acids, which was shown to require pyridoxal phosphate in the cases of alanine and... 

Scheme 19.11 Dynamic kinetic resolution of a-amino esters based on pyridoxal phosphate-catalyzed racemization and enzymatic ester hydrolysis.

---