A substrate-induced switch in the reaction mechanism of a thermophilic esterase - Kinetic evidences and structural basis

The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k(1), k(-1), k(2), and k(3)) along with activation energies (E-1, E-1, E-2, and E-3) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from k(cat) = k(3) with a short acyl chain substrate (p-nitrophenyl hexanoate) to k(cat) = k(2) with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyl- and alcohol-binding sites.