Racemates racemase enzymes

The reversal of this process could potentially occur with reprotonation from either face of the C=N double bond, and a mixture of aldimines would result, leading to generation of a racemic amino acid. This accounts for the mode of action of PLP-dependent amino acid racemase enzymes. Of course, the enzyme controls removal and supply of protons this is not a random event. One important example of this reaction is alanine racemase, employed by bacteria to convert L-alanine into o-alanine for cell-wall synthesis (see Box 13.12). 

Somewhat different conclusions were reached for mandelate racemase [38], Dissociation constants for the enzyme substrate complexes in the absence (k5) and presence (/c3) of Mn2+ were obtained (from proton relaxation rate titration of E-Mn2+ complex and other means). Evidence was obtained that in this case k3 was approximately equal to k4. Thus the authors concluded that the enzyme-catalyzed racemization steps should be approximately equal for both the D- and L-enantiomers , and termed this a rather startling prediction . Clearly, more detailed studies of the behavior of this, and other racemase enzymes would be desirable. 

Figure 12.7 Reaction profile of the enzymatic racemization of D- and L-5-monosubstituted hydantoin to the racemic mixture by hydantoin racemase enzyme monitored by chiral HPLC (see the methodology in [28]).

Garcia-Viloca, M., Gonzalez-Lafont, A. and Lluch, J.M. (2001). A QM/MM study of the racemization of vinylglycolate catalysis by mandelate racemase enzyme. J. Am. Chem. Soc. 123, 709-721... 

Methionine was first reported from casein in 1922 by Mueller. It is a limiting amino acid in the monogastric s feed and the addition of synthetic methionine in animal feed started from the 1950s. The addition of amino acids in the feed increases the nutritional quality and conversion efficiency of low protein feed and hence lowers the feed cost. Methionine is commercially produced by either chemical synthesis, enzymatic methods or microbial fermentation. Methionine has an advantage that it can be supplied to animal feed as a chemically produced racemate or a racemic mixture as the mammals are able to convert it to utilizable form with a methionine racemase enzyme. Chemical production uses harmful chemicals and production from protein hydrolysates requires several separation steps. Chemical synthesis produces a racemic mixture and is acetylated to produce L-methionine. Microbial fermentation overcomes these difficulties and has added advantages over the racemate that it helps optimal nutrient utihzation. 

Asano et al. have developed an approach for the synthesis of D-amino acids through DKR using a two-enzyme system [55]. They had previously reported the discovery of new D-stereospecific hydrolases that can be applied to KR of racemic amino acid amides to yield D-amino acids. Combination of a D-stereospedfic hydrolase with an amino acid amide racemase allows performing DKR of i-amino acid amides yielding enantiomerically pure D-amino acids in excellent yields (Figure 4.29). 

Racemic hydantoins result from the reaction of carbonyl compounds with potassium cyanide and ammonium carbonate or the reaction of the corresponding cyanohydrins with ammonium carbonate (Bucherer-Bergs reaction). Hydantoins racemize readily under basic conditions or in the presence of hydantoin racemase, thus allowing DKR (Figure 6.43). Hydantoinases (EC 3.5.2.2), either isolated enzymes or whole microorganisms, catalyze the hydrolysis of five-substituted... 

In many cases, the racemization of a substrate required for DKR is difficult As an example, the production of optically pure cc-amino acids, which are used as intermediates for pharmaceuticals, cosmetics, and as chiral synfhons in organic chemistry [31], may be discussed. One of the important methods of the synthesis of amino acids is the hydrolysis of the appropriate hydantoins. Racemic 5-substituted hydantoins 15 are easily available from aldehydes using a commonly known synthetic procedure (Scheme 5.10) [32]. In the next step, they are enantioselectively hydrolyzed by d- or L-specific hydantoinase and the resulting N-carbamoyl amino acids 16 are hydrolyzed to optically pure a-amino acid 17 by other enzymes, namely, L- or D-specific carbamoylase. This process was introduced in the 1970s for the production of L-amino acids 17 [33]. For many substrates, the racemization process is too slow and in order to increase its rate enzymes called racemases are used. In processes the three enzymes, racemase, hydantoinase, and carbamoylase, can be used simultaneously this enables the production of a-amino acids without isolation of intermediates and increases the yield and productivity. Unfortunately, the commercial application of this process is limited because it is based on L-selective hydantoin-hydrolyzing enzymes [34, 35]. For production of D-amino acid the enzymes of opposite stereoselectivity are required. A recent study indicates that the inversion of enantioselectivity of hydantoinase, the key enzyme in the... 

The reaction mechanism for glutamate racemase has been studied extensively. It has been proposed that the key for the racemization activity is that the two cysteine residues of the enzyme are located on both sides of the substrate bound to the active site. Thus, one cysteine residue abstracts the a-proton from the substrate, while the other detivers a proton from the opposite side of the intermediate enolate of the amino acid. In this way, the racemase catalyzes the racemization of glutamic acid via a so-called two-base mechanism (Fig. 15). 

Several hundred tons of L-methionine per year are produced by enzymatic conversion in an enzyme membrane reactor. An alternative approach is dynamic resolution, where the unconverted enantiomer is racemized in situ. Starting from racemic /V-acetyl-amino acid, the enantioselective L-acylase is used in combination with an TV-acyl-amino acid racemase to enable nearly total conversion of the substrate. 

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. 

In Section 5.03.6.2, a stereoselective synthesis of L-homophenylalanine from the racemic AAacetylated amino acid is described. The authors, however, found that substrate solubility limited the utility of this procedure. Having found an L-N-carbamoylase in Bacillus kaustophilus, they introduced the gene for this enzyme together with that for the N-acyl amino acid racemase from D. radiodurans into E. coli for coexpression. These cells, permeabilized with 0.5% toluene, were able to deliver L-homophenylalanine in 99% yield and were able to be used for multiple reaction cycles. 

After formation of the aldimine, numerous factors in the enzyme facilitate deprotonation of the a-carbon (Fig. 3, Step II). The lysine liberated by transimi-nation is utilized as a general base and is properly oriented for effective deprotonation [11]. Furthermore, the inductive effects of the ring system are tuned to increase the stabilization of the quinoid intermediate. For example, the aspartate group that interacts with the pyridyl nitrogen of the co enzyme promotes proto-nation to allow the ring to act as a more effective electron sink. In contrast, in alanine racemase, a less basic arginine residue in place of the aspartic acid is believed to favor racemization over transamination [12]. 

Mandelate racemase, another pertinent example, catalyzes the kinetically and thermodynamically unfavorable a-carbon proton abstraction. Bearne and Wolfenden measured deuterium incorporation rates into the a-posi-tion of mandelate and the rate of (i )-mandelate racemi-zation upon incubation at elevated temperatures. From an Arrhenius plot, they obtained a for racemization and deuterium exchange rate was estimated to be around 35 kcal/mol at 25°C under neutral conditions. The magnitude of the latter indicated mandelate racemase achieves the remarkable rate enhancement of 1.7 X 10, and a level of transition state affinity (K x = 2 X 10 M). These investigators also estimated the effective concentrations of the catalytic side chains in the native protein for Lys-166, the effective concentration was 622 M for His-297, they obtained a value 3 X 10 M and for Glu-317, the value was 3 X 10 M. The authors state that their observations are consistent with the idea that general acid-general base catalysis is efficient mode of catalysis when enzyme s structure is optimally complementary with their substrates in the transition-state. See Reference Reaction Catalytic Enhancement... 

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). 

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, enzymes that catalyze the hydrolysis of hydantoins 7-11,147). Because synthetic hydantoins are accessible by a variety of chemical syntheses, including Strecker reactions, enan-tioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, because hydantoins are easily racemized chemically or enzymatically by appropriate racemases, dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases have been reported 147). However, if asymmetric induction is poor or if inversion of enantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of L-methionine as part of a whole cell system E. coll) (Figure 22) 148). 

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). ... 

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... 

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... 

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. 

Like Esyn, the multienzyme Cysyn is unable to carry out racemization reactions because of a lack of an integrated racemase function [ 18,42]. Enzyme-bound... 

The alanine racemization catalyzed by alanine racemase is considered to be initiated by the transaldimination (Fig. 8.5).26) In this step, PLP bound to the active-site lysine residue forms the external Schiff base with a substrate alanine (Fig. 8.5, 1). The following a-proton abstraction produces the resonance-stabilized carbanion intermediates (Fig. 8.5, 2). If the reprotonation occurs on the opposite face of the substrate-PLP complex on which the proton-abstraction proceeds, the antipodal aldimine is formed (Fig. 8.5,3). The subsequent hydrolysis of the aldimine complex gives the isomerized alanine and PLP-form racemase. The random return of hydrogen to the carbanion intermediate is the distinguishing feature that differentiates racemization from reactions catalyzed by other pyridoxal enzymes such as transaminases. Transaminases catalyze the transfer of amino group between amino acid and keto acid, and the reaction is initiated by the transaldimination, followed by the a-proton abstraction from the substrate-PLP aldimine to form a resonance-stabilized carbanion. This step is common to racemases and transaminases. However, in the transamination the abstracted proton is then tranferred to C4 carbon of PLP in a highly stereospecific manner The re-protonation occurs on the same face of the PLP-substrate aldimine on which the a-proton is abstracted. With only a few exceptions,27,28) each step of pyridoxal enzymes-catalyzed reaction proceeds on only one side of the planar PLP-substrate complex. However, in the amino acid racemase... 

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