A detailed kinetic analysis of the oxidation of mono-substituted mandelates catalysed by L-(+)-mandelate dehydrogenase (L-MDH) from Rhodotorula graminis has been carried out to elucidate the role of the substrate in the catalytic mechanism. Values of K-m and k(cat) (25 degrees C, pH 7.5) were determined for mandelate and eight substrate analogues. Values of the activation parameters, Delta H-double dagger and Delta S-double dagger (determined over the range 5-37 degrees C), for mandelate and all substrate analogues were compensatory resulting in similar low values for free energies of activation Delta G(double dagger) (approx. 60 kJ.mol(-1) at 298.15 K) in all cases. A kinetic-isotope-effect value of 1.1+/-0.1 was observed using D,L-[2-H-2]mandelate as substrate and was invariant over the temperature range studied. The logarithm of k(cat.) values for the enzymic oxidation of mandelate and all substrate analogues (except 4-hydroxymandelate) showed good correlation with Taft's dual substituent constant (where = sigma(I)+0.64 sigma(R)(+)) and gave a positive reaction constant value, rho, of 0.36+/-0.07. This linear free-energy relationship was verified by analysing the data using isokinetic methods. These findings support the hypothesis that the enzyme-catalysed reaction proceeds via the same transition state for each substrate and indicates that this transition state is relatively nonpolar but has an electron-rich centre at the alpha-carbon position.
|Number of pages||6|
|Publication status||Published - 1 Feb 1994|