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Papers In Press, published online ahead of print November 15, 2003
J. Biol. Chem, 10.1074/jbc.M307738200
Submitted on July 17, 2003
Revised on November 13, 2003
Accepted on November 15, 2003

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

Giuseppina De Simone, Luigi Mandrich, Valeria Menchise, Valeria Giordano, Ferdinando Febbraio, Mosè Rossi, Carlo ;Pedone, and Giuseppe Manco

Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Napoli 80131

Corresponding Author: g.manco{at}ibp.cnr.it

The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at 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 (k1, k-1, k2 and k3), along with activation energies (E1, E-1, E2 and E3) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from kcat=k3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to kcat=k2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecansulphonyl chloride), structurally related to the latter substrate, 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 site


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