Investigation of a general base mechanism for ester hydrolysis in C-C hydrolase enzymes of the alpha/beta-hydrolase superfamily: a novel mechanism for the serine catalytic triad.
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 alpha/beta-hydrolase family. A reaction parameter (rho) 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.
Li
Jian-Jun
JJ
Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL.
Bugg
Timothy D H
TD
D001565:Benzoates / Q000378:metabolism; D001665:Binding Sites; D019117:Burkholderia / Q000201:enzymology; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D004950:Esterases / Q000737:chemistry / Q000378:metabolism; D004952:Esters / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D012694:Serine / Q000737:chemistry*; D013379:Substrate Specificity
D001565:Benzoates; D029968:Escherichia coli Proteins; D004952:Esters; D009596:Nitrophenols; D012694:Serine; C008642:4-nitrophenyl acetate; D006867:Hydrolases; D004950:Esterases; C022635:serine esterase; D008049:Lipase; C497337:MhpC protein, E coli