Mandelate proton abstraction

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... 

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.

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... 

B mandelate racemase Mg2+ proton abstraction to generate a carbanionic intermediate reprotonation of the intermediate... 

A number of epimerases act at carbon centers that are a to thioester linkages with coenzyme-A (CoA). These enzymes are similar to mandelate racemase, in that they employ a metal cofactor (Table 7.1), which is thought to stabilize an eno-late intermediate. They also belong to the VOC superfamily of enzymes, whose members all involve proton abstraction, proton transfer and metal cofactors that stabilize anionic intermediates [48-50]. 

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 degradation of mandelic acid by the bacterium Pseudomonas putida (Chapter 25) is initiated by mandalate racemase, another (a/(3)8-barrel protein.101 X-ray structures of bound inhibitors together with modeling suggest that the side chain of Lys 264 is the catalytic base that abstracts the a-H from S-mandelate (Fig. 13-5) and that the catalytic pair of His 297 and Asp 270 acts as proton donor, or, in the reverse direction, as catalytic... 

If Ke signifies the concentrations of enol and keto tautomers in a carboxylic acid, then pfCE is the difference between the pKa values of the a-protons of the keto tautomer and the hydroxyl group of the enol tautomer. pfCE, however, is also the difference in pfCa values between the a-protons and the proton of the carbonyl group of the carbonyl-protonated acid, that is deemed to be decisive (Gerlt, 1991) for the kinetics of abstraction of a proton, rather than the pfQ value of the substrate in solution. The pki of mandelic acid (15.4) links the pka of the a-proton of the keto tautomer (22.0) with the pK.d value of the enol tautomer (6.6). The pk, value also links the pka value of the a-proton with that of the carbonyl-bound proton of the protonated mandelic acid. If the pka value of the carbonyl-bound proton of the protonated mandelic acid is assumed to be about -8.0 then the pfQ value of the a-proton is about 7.4. This value matches well with the pka -values of Lys and His residues which have been assigned recently in the active center of mandelate race-mase, so electrophilic catalysis alone is able to explain the catalytic power of mandelate racemase. 

The reaction to be catalyzed poses problems for the enzyme [34]. The abstraction of a proton from an aliphatic carbon atom is generally difficult and slow. The plf of the carbon-bound a-proton of mandelic acid is 22.0 [55], while the p/f the a-proton of the mandelate anion (as for the phenylacetate anion) is approximately 29 [36, 37]. In spite of this, mandelate racemase increases the rate of the racemiza-tion reaction by a factor of 1.7 x 10 to approximately 1000 per second at 25 °C at pH 7 [57, 55]. Interactions of mandelate with enzyme, analogous to those with inhibitor (5)-atrolactate in which one carboxylate oxygen atom is coordinated to the magnesium ion and also hydrogen-bonded to the e-ammonium group of Lysi 64,... 

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