Mechanism and Catalytic Site Atlas

Introduction

Serine-dependent hydrolases catalyse a two-step reaction involving acylation and deacylation, each proceeding via a tetrahedral intermediates. Asp-His both act to activate the nucleophilic serine by providing a proton sink. His195 deprotonates the Ser9 hydroxyl group, concomitantly with the nucleophilic attack of the serine residue on the carbonyl carbon of the substrate, forming a tetrahedral intermediate. This is stabilised by the formation of an oxyanion hole formed by hydrogen bonds to the main chain amides of Ser9 and Gly42, and the side chain amide of Asn74. The positively charged His195 donates a proton to the first leaving group, resulting in the collapse of the tetrahedral intermediate and the formation of the covalent acyl-enzyme intermediate. An incoming water molecule is deprotonated by His195. The activated water molecule nucleophilically attacks the central carbonyl carbon of the ester bond, hydrolysing the ester bond. This results in the formation and subsequent collapse of a tetrahedral intermediate, leading to the release of the product. The enzyme is regenerated by reprotonation of Ser9 by His195.