Kinetics and thermodynamics of mandelate racemase catalysis.
Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, producing a rate enhancement exceeding 15 orders of magnitude. The rates of the forward and reverse reactions catalyzed by the wild-type enzyme and by a sluggish mutant (N197A) have been studied in the absence and presence of several viscosogenic agents. A partial dependence on relative solvent viscosity was observed for values of kcat and kcat/Km for the wild-type enzyme in sucrose-containing solutions. The value of kcat for the sluggish mutant was unaffected by varying solvent viscosity. However, sucrose did have a slight activating effect on mutant enzyme efficiency. In the presence of the polymeric viscosogens poly(ethylene glycol) and Ficoll, no effect on kcat or kcat/Km for the wild-type enzyme was observed. These results are consistent with both substrate binding and product dissociation being partially rate-determining in both directions. The viscosity variation method was used to estimate the rate constants comprising the steady-state expressions for kcat and kcat/Km. The rate constant for the conversion of bound (R)-mandelate to bound (S)-mandelate (k2) was found to be 889 +/- 40 s(-1) compared with a value of 654 +/- 58 s(-1) for kcat in the same direction. From the temperature dependence of Km (shown to equal K(S)), k2, and the rate constant for the uncatalyzed reaction [Bearne, S. L., and Wolfenden, R. (1997) Biochemistry 36, 1646-1656], we estimated the enthalpic and entropic changes associated with substrate binding (DeltaH = -8.9 +/- 0.8 kcal/ mol, TDeltaS = -4.8 +/- 0.8 kcal/ mol), the activation barrier for conversion of bound substrate to bound product (DeltaH# = +15.4 +/- 0.4 kcal/ mol, TDeltaS# = +2.0 +/- 0.1 kcal/ mol), and transition state stabilization (DeltaH(tx) = -22.9 +/- 0.8 kcal/ mol, TDeltaS(tx) = +1.8 +/- 0.8 kcal/ mol) during mandelate racemase-catalyzed racemization of (R)-mandelate at 25 degrees C. Although the high proficiency of mandelate racemase is achieved principally by enthalpic reduction, there is also a favorable and significant entropic contribution.[1]References
- Kinetics and thermodynamics of mandelate racemase catalysis. St Maurice, M., Bearne, S.L. Biochemistry (2002) [Pubmed]
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