Mandelate racemase inhibitors

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,... 

Fig. 27. Use of a motif to orient the magnesium octahedron in a protein (mandelate racemase) to facilitate a reaction [115]. The motif in Fig. 26b serves to orient the direction in which substrate (modeled in this crystal structure by an inhibitor) binds...

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... 

A variety of structural analogs of mandelate had earlier been tested as potential substrates or reversible inhibitors of the racemase. - Consequently, much was known about the minimal structural requirements for binding to the enzyme. It was clear that both an aromatic ring and a carboxylate group (or other similar anionic group) generally aided in this binding. Therefore, initial candidates for the affinity label all included both a phenyl and carboxylate substituent. 

Both frans-yff-phenylglycidate and a-phenylglycidate were separately examined as potential irreversible inhibitors by incubating 3 mM of each with a small amount of racemase (0.5 mM) at 30° and pH 7.0. After 1 hr, about 25% of the initial enzymic activity remained after incubation with trans-/S-phenyl ycidate. After only 30 min, enzyme incubated with the a-phenylglycidate was entirely inactive. After exhaustive dialysis to remove the a-phenylglycidate, racemase activity did not return. Preliminary studies also showed that the presence of mandelate itself slowed the inactivation process. 

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