The two preceding papers [Powers, V. M., Koo, C. W., Kenyon, G. L., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue); Neidhart, D. J., Howell, P. L., Petsko, G. A., Powers, V. M., Li, R., Kenyon, G. L., & Gerlt, J. A. (1991) Biochemistry (second paper of three in this issue)] suggest that the active site of mandelate racemase (MR) contains two distinct general acid/base catalysts: Lys 166, which abstracts the α-proton from (S)-mandelate, and His 297, which abstracts the α-proton from (R)-mandelate. In this paper we report on the properties of the mutant of MR in which His 297 has been converted to asparagine by site-directed mutagenesis (H297N). The structure of H297N, solved by molecular replacement at 2.2-Å resolution, reveals that no conformational alterations accompany the substitution. As expected, H297N has no detectable MR activity. However, H297N catalyzes the stereospecific elimination of bromide ion from racemic p-(bromomethyl)mandelate to give p-(methyl)-benzoylformate in 45% yield at a rate equal to that measured for wild-type enzyme; the unreacted p-(bromomethyl)mandelate is recovered as (R)-p-(hydroxymethyl)mandelate. At pD 7.5, H297N catalyzes the stereospecific exchange of the α-proton of (S)- but not (R)-mandelate with D2O solvent at a rate 3.3-fold less than that observed for incorporation of solvent deuterium into (S)-mandelate catalyzed by wild-type enzyme. The pD dependence of the rate of the exchange reaction catalyzed by H297N reveals a pKa of 6.4 in D2O, which is assigned to Lys 166. These observations provide persuasive evidence that the reaction catalyzed by MR does, in fact, proceed via a two-base mechanism in which Lys 166 abstracts the α-proton from (S)-mandelate and His 297 abstracts the α-proton from (R)-mandelate. Moreover, the facile exchange of solvent deuterium into (S)-mandelate catalyzed by H297N demonstrates the formation of a transiently stable intermediate. The rate of this exchange reaction and the measured pKa value of Lys 166 suggests that the pKa of the α-proton of (S)-mandelate in the active site of H297N is ≤15 in contrast to the value of 22 recently reported for mandelic acid in solution [Chiang, Y., Kresge, A. J., P. Pruszynski, P., Schepp, N. P., & Wirz, J. (1990) Angew. Chem. Int., Ed. Engl. 29, 792]. Since the rates of elimination of bromide ion from p-(bromomethyl)mandelate and of exchange of the α-proton of mandelate catalyzed by wild-type enzyme are nearly identical with those observed for the H297N, an intermediate is also presumed to lie on the reaction pathway for the racemization reaction catalyzed by wild-type enzyme. These studies demonstrate the power of site-directed mutagenesis in providing otherwise inaccessible detail about the mechanism of an enzyme-catalyzed reaction.
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