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.
Highlights
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
Kinetic constants for wild type EST2 and mutant M211S/R215L were measured in the presence of propan-2-ol, which was demonstrated to be a competitive inhibitor for the enzyme [9]
One simple way to get an idea about the reaction mechanism is to measure the pNP “burst,” which is supposed to occur when the enzyme is added to the reaction mixture before the onset of the catalytic reaction, provided the release of the acid/ester is slow compared with the release of the alcohol
Summary
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. On the basis of these structural data, EST2 variants with preferential specificity toward monoacyl esters with acyl chain length greater than eight carbon atoms were designed and generated by site-directed and saturation mutagenesis [9]. With the aim to better characterize the reaction mechanism of these enzymes and to give an explanation of the observed activation effect, these properties were further analyzed both at the kinetic and structural level. The three-dimensional structure of double mutant complexed with a long acyl chain sulfonyl derivative was solved providing a structural explanation of the enzyme kinetic behavior
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