SUBSTRATE-ANALOGS AS PROBES OF THE CATALYTIC MECHANISM OF L-MANDELATEDEHYDROGENASE FROM RHODOTORULA-GRAMINIS

A detailed kinetic analysis of the oxidation of mono-substituted mande lates catalysed by L-(+)-mandelate dehydrogenase (L-MDH) from Rhodotor ula graminis has been carried out to elucidate the role of the substra te 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 mandela te and all substrate analogues were compensatory resulting in similar low values for free energies of activation Delta G(double dagger) (app rox. 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 substra te and was invariant over the temperature range studied. The logarithm of k(cat.) values for the enzymic oxidation of mandelate and all subs trate analogues (except 4-hydroxymandelate) showed good correlation wi th Taft's dual substituent constant <(sigma)over bar> (where <(sigma)o ver bar> = sigma(I)+0.64 sigma(R)(+)) and gave a positive reaction con stant value, rho, of 0.36+/-0.07. This linear free-energy relationship was verified by analysing the data using isokinetic methods. These fi ndings support the hypothesis that the enzyme-catalysed reaction proce eds via the same transition state for each substrate and indicates tha t this transition state is relatively nonpolar but has an electron-ric h centre at the alpha-carbon position.