| Abstract
| - Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion ofthe two enantiomers of mandelic acid with remarkable proficiency, producing a rate enhancement exceeding15 orders of magnitude. The rates of the forward and reverse reactions catalyzed by the wild-type enzymeand by a sluggish mutant (N197A) have been studied in the absence and presence of several viscosogenicagents. A partial dependence on relative solvent viscosity was observed for values of kcat and kcat/Km forthe wild-type enzyme in sucrose-containing solutions. The value of kcat for the sluggish mutant wasunaffected by varying solvent viscosity. However, sucrose did have a slight activating effect on mutantenzyme efficiency. In the presence of the polymeric viscosogens poly(ethylene glycol) and Ficoll, noeffect on kcat or kcat/Km for the wild-type enzyme was observed. These results are consistent with bothsubstrate binding and product dissociation being partially rate-determining in both directions. The viscosityvariation method was used to estimate the rate constants comprising the steady-state expressions for kcatand kcat/Km. The rate constant for the conversion of bound (R)-mandelate to bound (S)-mandelate (k2) wasfound to be 889 ± 40 s-1 compared with a value of 654 ± 58 s-1 for kcat in the same direction. From thetemperature dependence of Km (shown to equal KS), k2, and the rate constant for the uncatalyzed reaction[Bearne, S. L., and Wolfenden, R. (1997) Biochemistry36, 1646−1656], we estimated the enthalpic andentropic changes associated with substrate binding (ΔH = −8.9 ± 0.8 kcal/mol, TΔS = −4.8 ± 0.8kcal/mol), the activation barrier for conversion of bound substrate to bound product (ΔH⧧ = +15.4 ± 0.4kcal/mol, TΔS⧧ = +2.0 ± 0.1 kcal/mol), and transition state stabilization (ΔHtx = −22.9 ± 0.8 kcal/mol,TΔStx = +1.8 ± 0.8 kcal/mol) during mandelate racemase-catalyzed racemization of (R)-mandelate at 25°C. Although the high proficiency of mandelate racemase is achieved principally by enthalpic reduction,there is also a favorable and significant entropic contribution.
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