Amino acid racemases cofactors

Among the numerous enzymes that utilize pyridoxal phosphate (PLP) as cofactor, the amino acid racemases, amino acid decarboxylases (e.g., aromatic amino acids, ornithine, glutamic acid), aminotransferases (y-aminobutyrate transaminase), and a-oxamine synthases, have been the main targets in the search for fluorinated mechanism-based inhibitors. Pharmaceutical companies have played a very active role in this promising research (control of the metabolism of amino acids and neuroamines is very important at the physiological level). 

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... 

Amino acid racemases are important for bacteria because they need D-alanine in the biosynthesis of cell walls. These enzymes require pyridoxal as the active cofactor. A racemization reaction starts with the aldimine complex between pyridoxal and an a-amino acid (Scheme 2.4). Deprotonation occurs at the a-carbon of amino acid, due to the electron-sink effect of pyridoxal. Reprotonation of the quinonoid intermediate at the opposite side provides the desired product (pathway a in Scheme 2.4). However, reprotonation may also take place at the C4 of pyridoxal (pathway b in Scheme 2.4). This kills the catalyst because one of its product, pyridoxamine, can no longer racemize an amino acid. 

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. 

Rudnick and Abeles purified proline racemase to 95% homogeneity from Clostridium sticklandii, and characterized it 92. The enzyme is composed of two identical subunits with a molecular weight of about 38000, and is independent of any cofactors or metals. Most amino acid racemases require pyridoxal 5 -phosphate, which labilizes the bond between the a-hydrogen and the chiral center by aldimine formation with the a-amino group of the substrate. However, PLP is not involved in the reaction of proline racemase acting on an a-imino acid. The enzyme also acts on 2-hydroxy-L-proline and 2-allo-hydroxy-D-proline although slowly they are epimer-ized at a rate of 2 and 5% of the rate of L-proline racemization, respectively. L-Proline and D-proline showed Km values of 2.9 and 2.5 mti, respectively1119. ... 

Figure I Schematic views of reactions involved in peptide biosynthesis. (1) Adenylate formation involving nucleophilic attack of the carboxyl group at the a-phosphate of the MgATP --complex with release of MgPP. - (2) aminoacylation of the pantetheine cofactor by formation of the thio-late anion, attack of the mixed anhydride, and release of AMP (3) tentative view of the peptide bond formation by nucleophilic attack of the aminoacyl-nitrogen at the preceding thioester-car-boxyl, involving deprotonaiion-protonation (4) epimerization of an aminoacyl-thioester, a reaction differing from those catalyzed by the well-characterized amino acid racemases. (Altered from Ref. 13. )...

Stein X Kluge B, Vater J, Franke P, Otro A, VI/ittmann-Liebold B. Gramicidin S synthetase 1 (phenylalanine racemase), a prototype of amino acid racemases containing the cofactor 4 -phosphopametheine. Biochem 1995 34 4633-4642. 

In certain bacteria there is a specific nutritional requirement for D-amino acids which are found as components of cell structures or antimetabolites. Bacteria normally meet this need by the conversion of L-amino acids to D-amino acids and in the case of alanine, methionine and tryptophan the evidence suggests that these reactions are directly catalysed by amino acid racemases which have a cofactor requirement for pyridoxal phosphate . An oxidation-reduction cofactor may also be a general feature of racemases of this class. However, the mode of epimerisation of L-phenylalanine to D-phenylalanine necessary for the synthesis of some peptide antibiotics, proceeds in an entirely different way, which as yet has only been partially resolved. 

We recently succeeded in determining the three-dimensional (3D) structure of AMDase by X-ray crystallography [16]. AMDase is structurally similar to cofactor-independent amino acid racemases [17, 18]. Considering the similarities of ligand... 

Perhaps the best characterized organic cofactor-dependent racemase is alanine racemase, which employs pyridoxal 5 -phosphate (PLP) (Table 7.1). o-alanine is necessary for the synthesis of the peptidoglycan layer of bacterial cell walls in Gram negative and positive bacteria [1]. Alanine racemase is thus a ubiquitous enzyme in bacteria and an excellent drug target [2]. Both its crystal structure and mechanism have been well investigated. PLP reacts with amino acids to produce... 

The presence of o-serine in mammalian brain tissue was first reported in 1989 [33, 34]. It has recently been established that o-serine is employed in the mammalian forebrain as a co-agonist for the N-methyl-o-asparate (NMDA) excitatory amino acid receptor [35, 36]. A PLP-dependent serine racemase has been cloned and purified from mammalian brain, and found to be a homodimer, which has a number of nonessential cofactors that enhance its activity, including Ca +, Mg + and ATP [37-40]. The mouse brain enzyme has also been shown to catalyze elimination from L-serine, to form pyruvate, with an activity comparable to that for racemiza-tion [41]. Interestingly, the first instance of this class of racemase was discovered by Esaki and coworkers in the silkworm, Bombyx mori [42]. o-serine concentration in the blood of B. mori larvae is thought to play a role in metamorphosis. 

Alanine racemase is a bacterial enzyme that catalyzes racemization of l- and d-alanine, and requires pyridoxal 5 -phosphate (PLP) as a cofactor. The enzyme plays an important role in the bacterial growth by providing D-alanine, a central molecule in the peptidoglycan assembly and cross-linking, and has been purified from various sources15 161. The enzyme has been used for the production of stereospecifically deuterated NADH and various D-amino acids by combination of L-alanine dehydrogenase (E. C. 1.4.1.1), D-amino acid aminotransferase (E. C. 2.6.1.21), and formate dehydrogenase (E.C. 1.2.1.2)I17, 18. ... 

Cofactor-independent Racemases and Epimerases Acting on Amino Acids... 

The synthesis of optically pure L-phenylglycine via the deracemization of mandelic acid was reported via three steps (racemization, enantioselective oxidation and stereoselective reductive amination). Racemization by mandelate racemase combined with simultaneous oxidation and reduction reactions with cofactor recycling gave the amino acid in 97% ee and 94% yield (Scheme 4.43) [96]. 

Racemases are enzymes capable of interconverting D- to L-amino acids. Pyridoxal phosphate has been claimed to play a role as a cofactor in bacterial racemases for alanine, glutamic acid, and methionine, but not in others. It has also been claimed that in mammals the administration of pyridoxine facilitates the use of D-amino acids. 

Scheme 12.2. The reversible transamination of aspartate (Asp, D) and alanine (Ala, A) as catalyzed by aminotransferase using pyridoxal as a cofactor. In principle (and apparently in practice), the same process can be used to convert any a-ketocarboxyhc acid to the corresponding amino acid. The a-proton is picked up and deposited on the same side (unless a racemase is involved) of the two amino acids. EC numbers and some graphic materials provided in this scheme have been taken from appropriate hnks in a URL starting with http // www.chem.qmul.ac.uk/iubmb/enzyme/.

The existence of enzymes in microorganisms which catalyze the interconversion of D- and L-amino acids is of considerable interest, since the intramolecular transfer of an amino group is apparently involved. The term racemase has been proposed for such enzymes. Two racemases have been reported. Alanine racemase has been shown to be present in a large number of microorganisms and has been partially purified from extracts of S. faecalis. Glutamic acid racemase has been demonstrated in acetone powders of Lactobacillus arabinosus. Both enzymes catalyze the interconversion of the n- and l- forms of their respective substrates. Alanine racemase requires pyridoxal phosphate as coenzyme. Pyri-doxamine phosphate under the conditions employed was not active. Glutamic acid racemase was found not to be affected by the addition of pyridoxal phosphate. However, further studies with purified preparations are necessary before pyridoxal phosphate can be excluded as cofactor for the glutamic acid racemase. Examination of animal tissues under conditions favorable for the demonstration of bacterial alanine racemase failed to reveal any activity. 

Racemases are enzymes that catalyze the inversion of the chiral center by deprotonation of the C , followed by reprotonation on the opposite face of the planar carban-ionic transition-state species [13,14], In order to overcome the high energetic barrier of racemization, for example, on a-amino acids, some racemases employ pyridoxal phosphate (PLP) as a cofactor to use the resonance-stabilized amino acid complex as an electron sink because the estimated pK values for the C of amino acids are high, in the range 21-32 [14,15]. The formation of an imine PLP-substrate covalent bond makes the pK value of a-hydrogen of amino acids low. The second class of enzymes includes proline, aspartate, and glutamate racemases and diaminopimelate epimer-ase, with a cofactor-independent two-base mechanism [14],... 

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