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J. Biol. Chem., Vol. 279, Issue 8, 6815-6823, February 20, 2004

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A Substrate-induced Switch in the Reaction Mechanism of a Thermophilic Esterase

KINETIC EVIDENCES AND STRUCTURAL BASIS^*

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

From the Istituto di Biostrutture e Bioimmagini-Consiglio Nazionale delle Ricerche, via Mezzocannone 6, 80134 Naples, Italy and the ^¶Istituto di Biochimica delle Proteine-Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy

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 k_cat = k₃ with a short acyl chain substrate (p-nitrophenyl hexanoate) to k_cat = 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 acyland alcohol-binding sites.

Received for publication, July 17, 2003 , and in revised form, November 13, 2003.

The atomic coordinates and structure factors (code 1QZ3) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by grants from the National Research Council of Italy (CNR) Target Project "PF-Biotechnology" and from the Regione Campania Legge 41/94 Year 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^|| Supported by a Ph.D. grant from the University Federico II of Naples.

** To whom correspondence may be addressed. Tel.: 39-081-2536651; Fax: 39-081-5514305; E-mail: pedone{at}chemistry.unina.it. To whom correspondence may be addressed. Tel.: 39-081-6132296; Fax: 39-081-6132248; E-mail: g.manco{at}ibp.cnr.it.

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