Racemization of alanine

Very detailed studies on the inhibition of alanine racemase by fluoroalanines have been conducted. This enzyme catalyzes the racemization of alanine to provide D-alanine, which is required for synthesis of the bacterial wall. This work has demonstrated that a more complex process than that represented in Figure 7.47 could intervene. For instance, in the case of monofluoroalanine, a second path (Figure 7.48, path b) occurs lysine-38 of the active site can also attack the Schiff base PLP-aminoacrylate that comes from the elimination of the fluorine atom. This enamine inactivation process (path b) has been confirmed by isolation and identification of the alkylation compound, after denaturation of the enzyme (Figure 7.48). ... 

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. 

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-... 

SHMT also catalyzes racemization of alanine and transamination of both its enantiomers. The particular reaction catalyzed by SHMT is mainly determined by the structure of the amino acid substrate. In the case of serine or glycine, the true substrates, SHMT does not catalyze any of the alternate reactions. The currently accepted model attributes this reaction specificity to the existence of open and closed active-site conformations. The physiological substrates generate the closed conformation, whereas alternate substrates react while the enzyme remains in the open conformation, which permits reaction paths leading to decarboxylation, transamination, and racemization. ... 

TPL also catalyzes the reverse reaction and may perform other 0 ,/3-elimination and /3-replacement reactions. In addition to L-tyrosine, L- and D-serine, 5-methyl-L-cysteine,. S-ethyl-L-cysteine, 5-(o-nitrophenyl)-L-cysteine, 0-benzoyl-L-serine, ° and /3-chloro-L-alanine also act as substrates for /3-elimination. TPL also catalyzes racemization of alanine. ... 

The improvements in resolution achieved in each deconvolution step are shown in Figure 3-3. While the initial library could only afford a modest separation of DNB-glutamic acid, the library with proline in position 4 also separated DNP derivatives of alanine and aspartic acid, and further improvement in both resolution and the number of separable racemates was observed for peptides with hydrophobic amino acid residues in position 3. However, the most dramatic improvement and best selectivity were found for c(Arg-Lys-Tyr-Pro-Tyr-(3-Ala) (Scheme 3-2a) with the tyrosine residue at position 5 with a resolution factor as high as 28 observed for the separation of DNP-glutamic acid enantiomers. 

OS 26] [R 4] [P 18] For dipeptide formation from the pentafluorophenyl ester of Boc-D-alanine and (S)-a-methylbenzylamine an extent of racemization of 5.6% was found [86]. This experiment also served to demonstrate monitoring of the racemization of an a-amino acid used in peptide synthesis. 

Jere et al. also reported on the stereoretentive C-H bond activation in aqueous phase catalytic hydrogenation of alanine (19). They demonstrated that hydrogenation of the carboxylic functionality is a stereo retentive process. Racemization occurs through a distinct process. 

B Liberek. The nitrile group in peptide chemistry. V. Racemization during peptide synthesis. 4. Racemization of active esters of phthaloyl-P-cyano-L-alanine in the presence of trie thy lamine. Acad Pol Sci Ser Sci Chim 11, 677, 1963. 

Kim s group in Seoul report the application of another of the E. coli ATs, the aromatic l-AAT encoded by the tyrB gene, to enrich the D-component of racemic preparations of alanine substituted at the /3-position with pyrazole, triazole, and imidazole. They also carried out an in silico investigation based on the crystal structure (PDB 3TAT) providing a reasonable rationalization (and therefore also potentially prediction) of substrate specificities. 

PLP-dependent enzymes catalyze the following types of reactions (1) loss of the ce-hydrogen as a proton, resulting in racemization (example alanine racemase), cyclization (example aminocyclopropane carboxylate synthase), or j8-elimation/replacement (example serine dehydratase) (2) loss of the a-carboxylate as carbon dioxide (example glutamate decarboxylase) (3) removal/replacement of a group by aldol cleavage (example threonine aldolase and (4) action via ketimine intermediates (example selenocysteine lyase). 

The first 3,6-dialkoxy-2,5-dihydropyrazine used in asymmetric synthesis of amino acids 7 10 was the symmetrical derivative 2, derived from cyclo(L-Ala, L-Ala) (1). This dihydropyrazine can be prepared by direct condensation of the methyl ester of L-alanine and subsequent alkylation with trialkyloxonium tetrafluoroborate7. Although the condensation process results in partial racemization of the alanine moiety, recrystallization yields almost optically pure cyclo(L-Ala, L-Ala) (1). 

Structure of the lithio derivatives (84CC853) The crystal structure of a THF solvate of the lithium derivative of racemic bislactim ether (derived from two molecules of alanine) has been determined. In the solid state, the lithium derivative exists as a dimer in which the two lithium atoms are nonequivalent (189). The two organic moieties in each dimer are homo-chiral this means that the crystal contains equal number of enantiomers. The Li Li distance is 2.61 A. In THF solution at - 108°C, the compound seems to exist as an equilibrium mixture of monomer and dimer in the ratio 5 1. It is not clear at the moment whether the reacting species is the monomer or the dimer. 

Racemization. A proton can be added back to the original alpha position but without stereospecificity. A racemase which does this is important to bacteria. They must synthesize D-alanine and D-glu-tamic acid from the corresponding L-isomers for use in formation of their peptidoglycan envelopes.153-1543 The combined actions of alanine racemase plus D-alanine aminotransferase, which produces D-glutamate as a product, provide bacteria with both d amino acids. 

The process of racemization is important in the survival and growth of living cells and is catalyzed by a group of enzymes called racemases. Alanine racemase. for example, is able to convert n-alanine to DL-alanine if a suitable alpha keto acid is also present. In this reaction the asymmetry of the alpha-carbon atom of alanine is lost as the amino acid is converted to the keto acid and back. This process is analogous to the well-known process of transamination in which racemization seldom occurs. 

A recent paper460 has questioned some of the above results and reports that Ni11 when complexed to L-alanine and Co111 when complexed to Gly-Ala or Ala-Gly retard the rate of racemization of the L-alanine. A detailed study of the pH rate profile on free and Nin-complexed L-alanine indicated that above pH 4 the free amino acid racemized faster than the complex. Further studies of these reactions are required. 

Fig. 8.6 Partial reaction profile of alanine racemization by B. stearothermophilus. Dotted lines indicate kinetically insignificant steps. (Reproduced with permission from Faraci and Walsh, Biochemistry, 27, 3264(1988)).

The favorable effect of the enamide function on asymmetric induction is indicated not only by the result with compound I, but also by later results summarized in Table I, where optical purities in the range of 70 to 80% were generally obtained for various derivatives of alanine, phenylalanine, tyrosine, and 3,4-dihydroxyphenylalanine (DOPA). The Paris group found that the Rh-(-)-DIOP catalyst yielded the unnatural R or d -amino acid derivatives, whereas l-amino acid derivatives could be obtained with a (+)-DIOP catalyst. Since the optical purity of the IV-acylamino acids can often be considerably increased by a single recrystallization (fractionation of pure enantiomer from racemate) and the IV-acetyl group can be removed by acid hydrolysis, this scheme provides an excellent asymmetric synthesis route to several amino acids. 

The optical purity of the iminazoline consisting of alanine and 2-aminopiperidine can be increased to 93.8 (e.e.)% by crystallizing it In Figure 14(a), once a crystal is removed to the outside of the system, it forms the solution in which asymmetric transformation is carried out the second-order asymmetric transformation where further transformation develops is obtained. In the case of crystallization of a dia-stereomer, 100 (e.e.)% might be possible. In this instance, the alanine appears to be racemized when the iminazoline ring is decomposed with strong acid. 

Racemization of Protein-Bound Amino Acids. D/L enantio-meric ratios for seven amino acid residues are given in Table I. Extensive racemization of aspartic acid, phenylala-nine, glutamic acid, and alanine occurred when the four proteins were treated with hydroxide. Valine, leucine, and proline were much less racemized. 

Alanine Racemase (EC 5.1.1.1) Alanine racemase is a PLP-dependent bacterial enzyme that catalyzes the racemization of L- and D-alanine exdusively (for a review see [60]). The enzyme from Salmonella typhimurium is an exception since it also accepts L-Ser, L-homo-Ser, and L-Cys as substrates. Alanine racemase, the most studied member of the PLP-dependent racemases, plays a major role in the bacterial growth by providing D-Ala for the peptidoglycan assembly and cross-Unking. 

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