De Simone_2004_J.Biol.Chem_279_6815

Reference

Title : A substrate-induced switch in the reaction mechanism of a thermophilic esterase: kinetic evidences and structural basis - De Simone_2004_J.Biol.Chem_279_6815
Author(s) : De Simone G , Mandrich L , Menchise V , Giordano V , Febbraio F , Rossi M , Pedone C , Manco G
Ref : Journal of Biological Chemistry , 279 :6815 , 2004
Abstract :

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 (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-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.

PubMedSearch : De Simone_2004_J.Biol.Chem_279_6815
PubMedID: 14617621
Gene_locus related to this paper: aliac-est2

Citations formats

De Simone G, Mandrich L, Menchise V, Giordano V, Febbraio F, Rossi M, Pedone C, Manco G (2004)
A substrate-induced switch in the reaction mechanism of a thermophilic esterase: kinetic evidences and structural basis
Journal of Biological Chemistry 279 :6815

De Simone G, Mandrich L, Menchise V, Giordano V, Febbraio F, Rossi M, Pedone C, Manco G (2004)
Journal of Biological Chemistry 279 :6815