MECHANISM OF THE REACTION CATALYZED BY MANDELATE RACEMASE - IMPORTANCE OF ELECTROPHILIC CATALYSIS BY GLUTAMIC-ACID-317

In the high-resolution X-ray structure of mandelate racemase (MR) with the competitive inhibitor (S)-atrolactate bound in the active site [L andro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., & Petsko, G. A. (1994) Bio chemistry 33, 635-643], the carboxylic acid group of Glu 317 is hydrog en-bonded to the carboxylate group of the bound inhibitor. This geomet ry suggests that the carboxylic acid functional group of Glu 317 parti cipates as a general acid catalyst in the concerted general acid-gener al base catalyzed formation of a stabilized enolic tautomer of mandeli c acid as a reaction intermediate. To test this hypothesis, the E317Q mutant of MR was constructed and subjected to high-resolution X-ray st ructural analysis in the presence of (S)-atrolactate. No conformationa l alterations were observed to accompany the E317Q substitution at 2.1 A resolution. The values for k(cat) were reduced 4.5 x 10(3)-fold for (R)-mandelate and 2.9 x 10(4)-fold for (S)-mandelate; the values for k(cat)/K-m were reduced 3 x 10(4)-fold. The substrate and solvent deut erium isotope effects measured for both wild-type MR and the E317Q mut ant are not multiplicative when deuteriated substrate is studied in D2 O, which suggests that the reactions catalyzed by both enzymes are ste pwise and involve the formation of stabilized enolic intermediates. In contrast to wild-type MR, E317Q does not catalyze detectable eliminat ion of bromide ion from either enantiomer of p-(bromomethyl)mandelate. However, E317Q is irreversibly inactivated by racemic alpha-phenylgly cidate at a rate comparable to that measured for wild-type MR. Taken t ogether, these mechanistic properties confirm the importance of Glu 31 7 as a general acid catalyst in the reaction catalyzed by wild-type MR . The k(cat) for wild-type MR and the reduction in k(cat) observed for E317Q are discussed in terms of the analysis recently described by Ge rlt and Gassman for understanding the rates and mechanisms of enzyme-c atalyzed proton abstraction reactions from carbon acids [Gerlt, J. A., & Gassman, P. G. (1993) J. Am. Chem. Soc. 115, 11552-11568; Gerlt, J. A., & Gassman, P. G. (1993) Biochemistry 32, 11943-11952].