A racemase

D-Methylmalonyl-CoA, the product of this reaction, is converted to the L-isomer by methylmalonyl-CoA epunerase (Figure 24.19). (This enzyme has often and incorrectly been called methylmalonyl-CoA racemase. It is not a racemase because the CoA moiety contains five other asymmetric centers.) The epimerase reaction also appears to involve a carbanion at the a-position (Figure 24.20). The reaction is readily reversible and involves a reversible dissociation of the acidic a-proton. The L-isomer is the substrate for methylmalonyl-CoA mutase. Methylmalonyl-CoA epimerase is an impressive catalyst. The for the proton that must dissociate to initiate this reaction is approximately 21 If binding of a proton to the a-anion is diffusion-limited, with = 10 M sec then the initial proton dissociation must be rate-limiting, and the rate constant must be... 

The main application of the enzymatic hydrolysis of the amide bond is the en-antioselective synthesis of amino acids [4,97]. Acylases (EC 3.5.1.n) catalyze the hydrolysis of the N-acyl groups of a broad range of amino acid derivatives. They accept several acyl groups (acetyl, chloroacetyl, formyl, and carbamoyl) but they require a free a-carboxyl group. In general, acylases are selective for i-amino acids, but d-selective acylase have been reported. The kinetic resolution of amino acids by acylase-catalyzed hydrolysis is a well-established process [4]. The in situ racemization of the substrate in the presence of a racemase converts the process into a DKR. Alternatively, the remaining enantiomer of the N-acyl amino acid can be isolated and racemized via the formation of an oxazolone, as shown in Figure 6.34. 

Racemic a-amino amides and a-hydroxy amides have been hydrolyzed enantio-selectively by amidases. Both L-selective and o-selective amidases are known. For example, a purified L-selective amidase from Ochrobactrum anthropi combines a very broad substrate specificity with a high enantioselectivity on a-hydrogen and a,a-disubstituted a-amino acid amides, a-hydroxyacid amides, and a-N-hydroxya-mino acid amides [102]. A racemase (a-amino-e-caprolactam racemase, EC 5.1.1.15) converts the o-aminopeptidase-catalyzed hydrolysis of a-amino acid amides into a DKR (Figure 6.38) [103]. 

Dynamic Kinetic Resolution Using an W-Acylase and a Racemase 85... 

Hsu et have cloned two enzymes from Deimcoccus radiodurans for overexpression in E. coli in order to carry out a dynamic kinetic resolution to obtain L-homophenylalanine, frequently required for pharmaceutical synthesis. The starting material is the racemic mixture of A acetylated homophenylalanine, and the two enzymes are an amino acid A -acylase, which specifically removes the acetyl group from the L-enantiomer, and a racemase, which interconverts the D- and L-forms of the A acyl amino acids. The resolution was carried out successfully using whole-cell biocatalysts, with the two enzymes either expressed in separate E. coli strains or coexpressed in the same cells. 

A class of enzymes that catalyze the interconversion of one enantiomer with its mirror image. Care must be exercised in applying this term. For example, the enzyme that interconverts D-methyhnalonyl-CoA to L-meth-ylmalonyl-CoA is not a racemase, but is instead an epi-merase the two coenzyme A derivatives are diastereo-isomeric, and not enantiomeric, with respect to each other. 

The racemization of an amino acid provides a biochemical example that can be related directly to Eq. 6-9. A solution of an L-amino acid will be efficiently changed into the racemic mixture of 50% d and 50% l by the action of an enzyme (a racemase) with no uptake or evolution of heat. Thus, AH = 0 and the only change is an entropy change. Let us designate 2 for the pure isomer as 2. Since there are just two choices of configuration for each of the N molecules in 1 mole of the racemate we see that for the racemate... 

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 plant acid S-mandelate must undergo conversion to R-mandelate by action of a racemase (Fig. 13-5) dehydrogenation, and side-chain cleavage as shown in Eq. 25-8 to form benzoate before it can be metabolized further.165... 

D-Alanine is found in bacterial cell wall peptidogly-can. l-Alanine is converted to D-alanine by a racemase that contains pyridoxal phosphate as a cofactor. The racemiza-tion is followed by the formation of a D-alanyl-D-alanine dipeptide, which is accompanied by the conversion of ATP to ADP. The dipeptide is subsequently incorporated into the glycopeptide (see fig. 16.16). 

After separation of the L-amino acid through ion exchange or by a crystallization step, the remaining N-acetyl-D-amino acid is recycled by thermal racemization under drastic conditions or by a racemase to achieve an overall yield of around 45% of L-amino acid (50% is the theoretical maximum) (Figure 7.11). D-Amino acids are also accessible by chemical hydrolysis of the N-acetyl-D-amino acid or directly by use of D-selective acylases. 

Kll. Kang, E. S., Snodgrass, P. J., and Gerald, P. S., Methylmalonic-coenzyme A racemase defect Another cause of methylmalonic aciduria. Pediatr. Res. 6, 875-879 (1972). 

In 1978 the conversion of the racemic 2-amino-d2-thiazoline-4-carboxylic acid into L-cysteine, catalysed by Pseudomonas thiazolinophilum cells, was reported [92]. Due to a racemase the overall process is a dynamic kinetic resolution and yields of 95% are obtained in the industrial process (Scheme 6.26) [34]. 

In elegant work the enantioselectivity of a hydantoinase from Arthrobader species for the production of l-methionine in Escherichia coli has been inverted [87]. The approach is similar to the one used in the evolution of (S)- and ( -selective lipases (see above). All known hydantoinases are selective for D-5-(2-methylthioethyl)hydantoin (d-18) which leads to the accumulation of N-carbamoyl-D-methionine (d-19), conversion being complete if the conditions of dynamic kinetic resolution are upheld [88], in this case by the use of a racemase or pH >8 (Fig. 11.22). 

In order to extend the two-enzyme system to other 2-hydroxy acids, a racemase with a broader activity was found in Lactobacillus paracasei. This was exploited for deracemization of 2-hydroxy-4-phenylbutanoic acid and 3-phenyllactic acid, which are important synthetic intermediates. In addition, in this case the procedure requires a kinetic resolution step and a successive racemization step. O-Acetyl derivatives of the absolute (S)-configuration can be obtained in two successive repeating cycles. Yields are around 60%. Of course the 0-acetyl derivatives of opposite configuration can be obtained when the lipase-catalyzed reaction is apphed in the hydrolysis direction. Obtaining the O-acetyl derivatives of the absolute (R)-configuration requires an additional acetylation step of the initially resolved and racemized (S)-hydroxy acid [12]. 

In addition, the use of a whole-cell biocatalyst consisting of a racemase, hydantoi-nase, and carbamoylase allows a dynamic biocatalytic resolution. Besides resolution processes, asymmetric (bio-)catalytic concepts have been applied successfully on an industrial scale. The different types of asymmetric (bio-)catalytic syntheses of L-amino acids, based on the use of prochiral starting materials, are shown in Fig. 3. 

The reaction concept with this new hydantoinase-based biocatalyst is economically highly attractive since it represents a dynamic kinetic resolution process converting a racemic hydantoin (theoretically) quantitatively into the enantiomerically pure L-enantiomer [19]. The L-hydantoinase and subsequently the L-carbamoylase hydrolyze the L-hydantoin, l-11, enantioselectively forming the desired L-amino acid, l-2. In addition, the presence of a racemase guarantees a sufficient racemiza-tion of the remaining D-hydantoin, d-11. Thus, a quantitative one-pot conversion of a racemic hydantoin into the desired optically active a-amino acid is achieved. The basic principles of this biocatalytic process in which three enzymes (hydan-toinase, carbamoylase, and racemase) are integrated is shown schematically in Fig. 9. 

Kundu UR, Li W, Tan D, et al. Alpha-methylacyl-co-a-racemase (AMACR) expression is a biomarker of poor prognosis in stage 1 and 2 large cell carcinomas of the lung. Mod Pathol. 2008 21 345A. 

Alpha-methylacyl coenzyme A racemase-reactivity has also been observed in EPD. However, that enzyme is potentially present in SCC and melanoma as welD and therefore has lesser diagnostic value compared with the aforementioned determinants. 

Mayes DC, Patterson JW, Ramnani DM, Mills SE. Alpha-methylacyl coenzyme A racemase is immunoreactive in extramammary Paget disease. Am Clin Pathol. 2007 127 567-571. 

Nasser A, Amin MB, Sexton DG, Cohen C. Utility of alpha-methylacyl coenzyme A racemase (p504S antibody) as a diagnostic immunohistochemical marker for cancer. Appl Im-munohistochem Mol Morphol. 2005 13 252-255. 

Huang W, Zhao), Li L, et al. a-Methylacyl coenzyme A racemase is highly expressed in the intestinal-type adenocarcinoma and high-grade dysplasia lesions of the stomach. Histol Histopathol. 2008 23 1315-1320. 

Chen ZM, Ritter JH, Wang HL. Differential expression of alpha-methylacyl coenzyme A racemase in adenocarcinomas of the small and large intestines. Am J Surg Pathol. 2005 29 890-896. 

Marx A, Wandrey T, Simon P, et al. Combined alpha-methylacyl coenzyme A racemase/p53 analysis to identify dysplasia in inflammatory bowel disease. Hum Pathol. 2009 40 166-173. 

Suh N, Yang XJ, Tretiakova MS, et al. Value of CDX2, villin, and alpha-methylacyl coenzyme A racemase immunostains in the distinction between primary adenocarcinoma of the bladder and secondary colorectal adenocarcinoma. Mod Pathol. 2005 18 1217-1222. 

TABLE 16.2 Immunoreactivity of a-Methylacyl Coenzyme A Racemase In Benign and Neoplastic Prostate ... 

FIGURE 16.7 Foci of partial atrophy (A and B) showing patchy basal cell layer positivity with high-molecular-weight cytokera-tin (C) and p63 (D). Partial atrophy glands may display positive a-methylacyl coenzyme A racemase staining (E). 

---