Pyridoxal-5 -phosphate isomers

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... 

This reaction is crucial because it establishes the stereochemistry of the a-carbon atom (S absolute configuration) in glutamate. The enzyme binds the a-ketoglutarate substrate in such a way that hydride transferred from NAD(P)H is added to form the 1 isomer of glutamate (Figure 24,6). As we shall see, this stereochemistry is established for other amino acids by transamination reactions that rely on pyridoxal phosphate. 

Tn amino acid production, we encounter an important problem in bio.synthesis—namely, stereochemical control. Because all amino acids except glycine are chiral, biosynthetic pathways must generate the correct isomer with high fidelity. In each of the 19 pathways for the generation ofchiral amino acids, the stereochemistry at the a-carbon atom is established by a transamination reaction that includes pyridoxal phosphate (PEP). Almost all the transaminases that catalyze these reactions descend from a common ancestor, illustrating once again that effective solutions to biochemical problems are retained throughout evolution. 

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. 

Bacterial Amino Acid Decarboxylases. Tyrosine decarboxylase was used extensively in studies on the nature of the amino acid decarboxylases. This activity is found in several microorganisms and the enzyme was purified over 100-fold from S. faecalis. It is easily resolved, and the apoenzyme has been used to assay pyridoxal phosphate (PALPO). Final concentrations of PALPO between 10 M and 10 M are conveniently measured by the rate of CO2 liberation from tyrosine. Phenylalanine is decarboxylated by this or a similar enzyme. Tyrosine decarboxylase and all of the other amino acid decarboxylases described in this section act only on the L-isomers of their respective substrates. 

Diaminopimelic acid is decarboxylated by a specific enzyme that requires pyridoxal phosphate. Only the meso isomer is attacked, and the product is L-lysine. L,ii-Diaminopimelic has been found to yield lysine in some systems this observation has been explained as caused by a race-mase that forms the meso compound. ... 

While this work was proceeding, Yanofsky and Reissig independently found the L-serine deaminase of Neurospora This enzyme is activated by pyridoxal phosphate, and is not stimulated by ATP, biotin, or glutathione. The Neurospora enzyme is somewhat more active with threonine than with serine, and acts only on the L-isomers. 

Both isomers of both serine and threonine are deaminated by Neuro-spora extracts 225-228). The enzymes involved have been purified by Yanofsky and his associates 225-228). A specific D-serine and D-threonine dehydrase has been purified thirty-five- to fortyfold from this mold 2f ). An absolute requirement for pyridoxal phosphate was demonstrated. No requirement for AMP or glutathione could be demonstrated. Some indication of a metal requirement was observed. The preparation was not active with the li-isomers of serine and threonine or DL-homoserine and DL-homo-cysteine. The rate of deamination of D-threonine is very slow compared to that with L-serine. Activity was observed with D-glutamic acid and D-as-partic acid. Since other D-amino acids were not deaminated by the preparation, these results could not be due to a contamination with D-amino acid oxidase. Furthermore, when either of these amino acids was incubated in the presence of D-serine, the keto acid production was a summation of that for each substrate alone. Pyridoxal phosphate had no effect on keto acid formation from the dicarboxylic amino acids. It is of interest that D-amino acid oxidase of Neurospora does not attack D-serine or D-threonine (77). 

The enzyme is active on both L-kynurenine and L-3-hydroxykynurenine. The D-isomers are inert. It requires pyridoxal phosphate as a coenzyme. The most effective amino-group acceptor is a-ketoglutarate, but other a-keto acids can also function. o-Aminobenzoylpyruvic acid, the expected product of the action of kynurenine transaminase on kynurenine, has not... 

Linatine was originally isolated as an vitamin Bs antagonist from seeds of Linum usitatissimm (flax seeds). The compound is toxic towards both chickens and Azotobacter species. 1-Amino-D-proline has the same toxic effect (158). The toxicity is apparently due to formation of the hydrazone of pyridoxal 5 -phosphate (296, 341). 1-Amino-L-proline and 1-amino-D-proline were found to be equally toxic towards chickens, but the D-isomer was 50 times more toxic than the L-isomer towards Azotobacter vinelandii (158). A review on naturally occurring Be antagonists has been published (157). 

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