Mechanism of the Reaction Catalyzed by Mandelate Racemase: Structure and Mechanistic Properties of the K166R Mutant

Abstract

On the basis of the available high-resolution structures of mandelate racemase (MR) from Pseudomonas putida [Landro, 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) Biochemistry 33, 635-643], Lys 166 and His 297 are positioned appropriately to participate in catalysis as acid/base catalysts that either abstract the alpha-proton from the enantiomers of mandelate to form an enolic intermediate or protonate the enolic intermediate to form the enantiomers of mandelate, with Lys 166 participating as the (S)-specific acid/base catalyst and His 297 participating as the (R)-specific acid/base catalyst. In this paper we report the structural and mechanistic properties of the mutant in which Lys 166 has been replaced with arginine (K166R). The structure of K166R has been determined at 1.85 A resolution with the substrate (S)-mandelate bound in the active site. The structure of this complex reveals no geometric alterations in the active site, with the exception that the longer side chain of Arg 166 is necessarily displaced upward from the position occupied by Lys 166 by steric interactions with the bound substrate. In contrast to the H297N mutant of MR [Landro, J. A., Kallarakal, A. T., Ransom, S. C., Gerlt, J. A., Kozarich, J. W., Neidhart, D. J., & Kenyon, G. L. (1991) Biochemistry 30, 9275-9281], the K166R exhibits low levels of racemase activity [kcat is reduced 5 x 10(3)-fold in the (R)- to (S)-direction and 1 x 10(3)-fold in the (S)- to (R)-direction]. The substrate and solvent deuterium isotope effects support a reaction coordinate for the K166R-catalyzed reaction in which the transition state for interconversion of bound (S)-mandelate and the stabilized enolic intermediate is higher in energy that the transition state for interconversion of bound (R)-mandelate and the stabilized enolic intermediate. The solvent deuterium isotope effect when (S)-mandelate is substrate (2.2 +/- 0.3) supports the proposal that the formation of the enolic intermediate involves partial transfer of a solvent-derived proton from Glu 317 to the substrate as the alpha-proton is abstracted [Mitra, B., Kallarakal, A. T., Kozarich, J. W., Gerlt, J. A., Clifton, J. G., Petsko, G. A., & Kenyon, G. L. (1995) Biochemistry 34, 2777-2787].(ABSTRACT TRUNCATED AT 400 WORDS)

Computer Simulation of Primary Kinetic Isotope Effects in the Proposed Rate-limiting Step of the Glyoxalase I Catalyzed Reaction

“…This type of mechanism is analogous to that of several other enzymes catalyzing reactions of carbon acids, such as triosephosphate isomerase (3), enolase (4), and mandelate racemase (5). It is thus of interest to elucidate possible common features and principles of enzyme catalysis for this class of reactions.…”

The Enolase Superfamily: A General Strategy for Enzyme-Catalyzed Abstraction of the α-Protons of Carboxylic Acids

“…E317 is the general acid catalyst that evidently donates a proton to the carboxyl group of the substrate as the R-proton is abstracted (not given in Table 2). These functions have been verified by site-directed mutagenesis, X-ray crystallography, and mechanistic analyses of mutant proteins (Landro et al, 1991;Mitra et al, 1995;Kallarakal et al, 1995;Schafer et al, 1996). The required Mg 2+ is coordinated to the carboxylate groups of D195, E221, and E247 as well as to one carboxylate oxygen and the R-hydroxyl group of the bound inhibitor.…”

Absolute Rate Calculations. Proton Transfers in Solution

“…121 In view of the slowness of the uncatalysed exchange with deuterium at pD 7.5, the effect of temperature on the first-order rate constant was determined at temperatures between 130 and 190°C. 92 The experimental rates of the uncatalyzed exchange are compared in Figure 7 with the rates calculated using the acidity constant of mandelic acid ionizing as a carbon acid, pK a K ) 22, 122,123 and the data for acetone and water.…”