| dc.description | Mandelate racemase (MR) (E.C. 5.1.2.2.) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid. The enzyme is remarkable in its ability to catalyze rapid carbon-hydrogen bond cleavage from a carbon acid with a high pKa, making it a useful paradigm for understanding enzyme-catalyzed proton abstraction from carbon acids. The proficiency of an enzyme catalyst is a function of its ability to provide greater stabilizing interactions with the altered substrate in the transition state than with the substrate in the ground state. To better understand the molecular origins of the high proficiency of MR, the relative contributions from enzyme binding determinants to the stabilization of the substrate in the ground state and the altered substrate in the transition state have been investigated. The rates of the forward and reverse reactions catalyzed by MR were dependent on the concentration of viscosogenic agents, demonstrating that both substrate binding and product dissociation are partially rate-determining in both directions. Using these viscosity studies over a range of temperatures, the enthalpic and entropic contributions to MR-catalyzed racemization of (R)-mandelate at 25°C were estimated. The high proficiency of MR was shown to be achieved principally through 23 kcal/mol of enthalpic reduction, suggesting that enzyme residues play a critical role in transition state stabilization by providing specific interactions with the altered substrate in the transition state. Investigation of the molecular origins of these interactions required the identification of a high affinity, intermediate analogue inhibitor to act as a stable probe for the enzyme-transition state complex. A fixed-time, high-performance liquid chromatography assay for MR activity was developed and validated. Using this assay and a circular dichroism-based assay, a series of carboxylate-, phosphonate- and hydroxamate-containing substrate and intermediate analogues were examined for their ability to inhibit MR. (Abstract shortened by UMI.) | en_US |
