Racemization of Vinylglycolate Catalyzed by Mandelate Racemase

Vinylglycolate (2-hydroxy-3-butenoic acid, 2) has been found to be an excellent substrate of mandelate racemase. The measured steady-state kinetic parameters for the enantiomers of 2 are comparable, with a maximal racemization rate that is 35% relative to mandelate. Racemization of 2 is subject to a primary deuterium kinetic isotope of about 4, indicating that abstraction of the α-proton is at least partially rate-limiting. Although α-hydroxybutyrate (5), the saturated analogue of 2, is not a substrate, 5 competitively inhibits racemization of 2 with a K_i value comparable to the average K_m value for the latter. These results implicate the importance of β,γ-unsaturation in promoting facile racemization of the substrate α-proton. In addition, the enzyme catalyzes the isomerization of 2 to α-ketobutyrate (4), with a partition ratio for racemization/isomerization of about 1 × 10⁴. These observations highlight the precision with which mandelate racemase can promote racemization to the virtual exclusion of a thermodynamically more favored process.