Enzyme mandelate racemase

Figure 4.9 Mechanisms of the reactions catalyzed by the enzymes mandelate racemase (a) and muconate lactonizing enzyme (b). The two overall reactions are quite different a change of configuration of a carbon atom for mandelate racemase versus ring closure for the lactonizing enzyme. However, one crucial step (red) in the two reactions is the same addition of a proton (blue) to an intermediate of the substrate (red) from a lysine residue of the enzyme (E) or. In the reverse direction, formation of an intermediate by proton abstraction from the carbon atom adjacent to the carboxylate group.

The enzyme mandelate racemase from the soil bacterium Pseudomonas putida catalyzes the racemization of the (/ )- and (5)-enantiomers of mandelate, as shown in Figure 3. In the action of the enzyme a hydrogen atom (a-proton) is extracted from the aliphatic carbon atom of the mandelate ion. Two active-site bases are involved in this one to extract the a-proton from the (5)-isomer and the other to extract the a-proton from the (R)-isomer [26]. A proton from a solvent molecule in the active site then adds to the deprotonated mandelate in one or other of the two... 

Figure 3. Interconversion of (/ )- and (S)-mandelate by the action of the enzyme mandelate racemase. The addition of a proton to one or other of the two faces of...

Figure 5. The locations of proton-abstracting groups in the enzyme mandelate racemase (PDB file 1MDR). The binding of (5)-atrolactate, which has an additional methyl group that causes it to inhibit the reaction of mandelate racemase, is shown. Presumably when mandelate is bound, one of the bases (Lys166 or His297) approaches the a-carbon atom of the mandelate ion.

The enzyme mandelate racemase interconverts the enantiomers of mandelate ion (2-hydroxyphenylacetic acid). 

These results are compatible with an evolutionary history in which the new enzyme activity of mandelate racemase has evolved from a preexisting enzyme that catalyzes the basic chemical reaction of proton abstraction and formation of an intermediate. Subsequent mutations have modified the... 

Neidhart, D.J., et al. Mandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous. Nature 347 ... 

Beanie, S. L., and Wolfenden, R., 1997. Mandelate racemase in pieces Effective concentrations of enzyme fnnctional groups in the transition state. Biochemistry 36 1646-1656. 

The mandelate pathway in Pseudomonas putida involves successive oxidation to benzoyl formate and benzoate, which is further metabolized via catechol and the 3-ketoadipate pathway (Figure 8.35a) (Hegeman 1966). Both enantiomers of mandelate were degraded through the activity of a mandelate racemase (Hegeman 1966), and the racemase (mdlA) is encoded in an operon that includes the next two enzymes in the pathway—5-mandel-ate dehydrogenase (mdlB) and benzoylformate decarboxylase (mdlC) (Tsou et al. 1990). 

Other Proteins The ouabain-binding site on (Na /K -adenosine-5 -triphosphatase, 46, 523 penicillin isocyanates for /3-lactamase, 46, 531 active site-directed addition of a small group to an enzyme the ethylation of ludferin, 46, 537 mandelate racemase, 46, 541 d imethylpyrazole carboxamidine and related derivatives, 46, 548 labeling of catechol O-methyltransferase with N-haloace-tyl derivatives, 46, 554 affinity labeling of binding sites in proteins by sensitized photooxidation, 46, 561 bromocolchicine as a iabei for tubuiin, 46, 567. 

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... 

Figure 13-5 An S-mandelate ion in the active site of mandelate racemase. Only some of the polar groups surrounding the active site are shown. The enzyme has two catalytic acid-base groups. Lysine 166 is thought to deprotonate S-mandelate to form the aci anion, while His 297 deprotonates R-mandelate to form the same anion.106...

G. L. Kenyon, J. A. Gerlt, G. A. Petsko, and J. W. Kozarich, Mandelate racemase structure-function studies of a pseudo-symmetric enzyme, Acc. Chem. Res. 1995, 28, 178-186. 

The mandelate and jS-ketoadipate pathways serve as an example of gene duplication, as there is strong evidence pointing to the former evolving from the latter. Evolution of mandelate racemase from muconate lactonizing enzyme points to the relevance of the enzyme mechanism for catalytic reactivity. 

Enolases mandelate racemase (MR), muconate-lactonizing enzyme (MLE), N-acetylamino acid racemase (NAAR) Hasson, 1998 Palmer, 1999... 

Mandelate racemase (MR) enables some strains of the common soil bacterium Pseudomonas putida to utilize mandelate from decomposing plant matter as a carbon source. MR is the first of five enzymes in the bacterial pathway that converts mandelate to benzoate. Then benzoate is broken down by another set of five enzymes of the ensuing /l-ketoadipate pathway to compounds that can be used to generate ATP, the cell s major source of chemical energy. 

The enolase superfamily story started with the serendipitous discovery that two enzymes catalyzing very different overall reactions, mandelate racemase (MR) and muconate lactonizing enzyme (MLE), had virtually superimposable structures (Neidhart et al., 1990). As shown in Figure 2, MR catalyzes the reversible racemization of mandelate, an aromatic substrate, while MLE catalyzes the equilibration of muconolactone with as, m-muconate. Given the substantial differences in these reactions, it... 

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. 

Deracemization of mandelic add with the combined action of two enzymes has been reported. rac-MandeUc acid is acylated by a Pseudomonas sp. lipase in diisopropyl ether. After solvent removal the mfacture of mandeUc acid enriched in the R-form and the 0-acetyl derivative of the S-configuration are subjected to the mandelate racemase-catalyzed racemization in aqueous buffer. In these conditions only the non-acetylated hydroxy acid is racemized. In order to obtain (S)-0-acetylmandelic acid in an 80% isolated yield and a >98% e.e. the process must be repeated four times [9]. 

Mandelate racemase, which is probably the best known of all racemizing enzymes [10], shows an activity limited to compounds structurally related to man-delates [11]. The presence of an aromatic group or an unsaturation in the a-position is essential for activity. 

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... 

The first example of the use of a two-enzyme system in ionic liquids was reported by Kaftzik et al. [75], who investigated the deracemization of ( )-mandelic acid using a lipase-mandelate racemase two-enzyme system in ionic hquids (Fig. 7.9). They used a combination of the mandelate racemase-catalysed racemisation of (R)-mandelic acid and the lipase-catalysed kinetic resolution of (5)-mandelic acid to... 

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