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

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

Authors :
Giuseppina, De Simone
Luigi, Mandrich
Valeria, Menchise
Valeria, Giordano
Ferdinando, Febbraio
Mosè, Rossi
Carlo, Pedone
Giuseppe, Manco
Source :
The Journal of biological chemistry. 279(8)
Publication Year :
2003

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.

Details

ISSN :
00219258
Volume :
279
Issue :
8
Database :
OpenAIRE
Journal :
The Journal of biological chemistry
Accession number :
edsair.pmid..........e6759ffb9d9358b2b92a584fe839254f