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Issue 3, 2007
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Investigation of a general base mechanism for esterhydrolysis in C–C hydrolase enzymes of the α/β-hydrolase superfamily: a novel mechanism for the serine catalytic triad

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Abstract

Previous mechanistic and crystallographic studies on two C–C hydrolase enzymes, Escherichia coli MhpC and Burkholderia xenovorans BphD, support a general base mechanism for C–C hydrolytic cleavage, rather than the nucleophilic mechanism expected for a serine hydrolase. The role of the active site serine residue could be to form a hydrogen bond with a gem-diolate intermediate, or to protonate such an intermediate. Hydrolase BphD is able to catalyse the hydrolysis of p-nitrophenyl benzoate ester substrates, which has enabled an investigation of these mechanisms using a Hammett analysis, and comparative studies upon five serine esterases and lipases from the α/β-hydrolase family. A reaction parameter (ρ) value of +0.98 was measured for BphD-catalysed ester hydrolysis, implying a build-up of negative charge in the transition state, consistent with a general base mechanism. Values of +0.31–0.61 were measured for other serine esterases and lipases, for the same series of esterase substrates. Pre-steady state kinetic studies of ester hydrolysis, using p-nitrophenyl acetate as the substrate, revealed a single step kinetic mechanism for BphD-catalysed ester hydrolysis, with no burst kinetics. A general base mechanism for BphD-catalysed ester hydrolysis is proposed, in which Ser-112 stabilises an oxyanion intermediate through hydrogen bonding, and assists the rotation of this oxyanion intermediate via proton transfer, a novel reaction mechanism for the serine catalytic triad.

Graphical abstract: Investigation of a general base mechanism for ester hydrolysis in C–C hydrolase enzymes of the α/β-hydrolase superfamily: a novel mechanism for the serine catalytic triad

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Article information


Submitted
27 Oct 2006
Accepted
27 Nov 2006
First published
19 Dec 2006

Org. Biomol. Chem., 2007,5, 507-513
Article type
Paper

Investigation of a general base mechanism for ester hydrolysis in C–C hydrolase enzymes of the α/β-hydrolase superfamily: a novel mechanism for the serine catalytic triad

J. Li and T. D. H. Bugg, Org. Biomol. Chem., 2007, 5, 507
DOI: 10.1039/B615605C

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