Mechanism of the Reaction Catalyzed by Mandelate Racemase. 3. Asymmetry in Reactions Catalyzed by the H297N Mutant

James A. Landro, Abraham T. Kallarakal, Stephen C. Ransom, John A. Gerlt, John W. Kozarich, David J. Neidhart, George L. Kenyon

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)9274-9281
Number of pages8
JournalBiochemistry
Volume30
Issue number38
DOIs
StatePublished - Sep 1 1991
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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    Landro, J. A., Kallarakal, A. T., Ransom, S. C., Gerlt, J. A., Kozarich, J. W., Neidhart, D. J., & Kenyon, G. L. (1991). Mechanism of the Reaction Catalyzed by Mandelate Racemase. 3. Asymmetry in Reactions Catalyzed by the H297N Mutant. Biochemistry, 30(38), 9274-9281. https://doi.org/10.1021/bi00102a020