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Papers In Press, published online ahead of print July 10, 2001
J. Biol. Chem, 10.1074/jbc.M103017200
Submitted on April 5, 2001
Revised on July 5, 2001
Accepted on July 9, 2001
Istituto di Biochimica delle Proteine ed Enzimologia, Consiglio Nazionale delle Ricerche, Napoli 80125
Corresponding Author: manco{at}dafne.ibpe.na.cnr.it
The recently solved three-dimensional structure of the thermophilic esterase 2 from Alicyclobacillus acidocaldarius allowed us to have a snapshot of an enzyme-sulphonate complex, which mimics the second stage of the catalytic reaction, namely the covalent acyl-enzyme intermediate. The aim of this work was to design by structure-aided analysis and to generate by site-directed and saturation mutagenesis, EST2 variants with changed substrate specificity in the direction of preference for monoacylesters whose acyl-chain length is greater than eight carbon atoms. Positions 211 and 215 of the polypeptide chain were chosen to introduce mutations. Among five variants with single and double amino acid substitutions, three were obtained, M211S, R215L, and M211S/R215L, which changed the catalytic efficiency profile in the desired direction. Kinetic characterization of mutants and wild type showed that this change was achieved by an increase in kcat and a decrease in Km values with respect to the parental enzyme. The M211S/R215L specificity constant for p-nitrophenyl decanoate substrate was 6-fold higher than the wild type. However, variants M211T, M211S, M211V showed strikingly increased activity as well as maximal activity with monoacylesters with 4 carbon atoms in the acyl chain, compared with the wild type. In the case of mutant M211T, the kcat for p-nitrophenyl butanoate was 2.4-fold higher. Overall, depending on the variant and on the substrate, we observed improved catalytic activity at 70°C with respect to the wild type, which was a somewhat unexpected result for an enzyme with already high kcat values at high temperature. In addition, variants with altered specificity toward the acyl-chain length were obtained. The results were interpreted in the context of the EST2 three-dimensional structure and a proposed catalytic mechanism in which kcat, e.g. the limiting step of the reaction, was dependent on the acyl chain length of the ester substrate.
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