Mechanism and Catalytic Site Atlas

Introduction

The Mpro active site is situated in a cleft between domains I (residues 8-101) and II (residues 102-184) and comprises a catalytic dyad consiting of conserved residues Cys 3385 and His 3281. A buried water molecule is hydrogen bonded to His 3281 and is considered the third component of a catalytic triad. The fold of the Mpro enzyme at the catalytic site resembles that of the serine proteinase, chymotrypsin, but with an active-site serine-to-cysteine substitution. The catalytic site contains a hydrogen bonded thiol....imidazole ion pair , with the uncharged thiol acting as the nucleophile, and the His 3821 as a general base. Deprotonation of the Cys 3385 sulfhydryl by an adjacent residue with a basic side chain. The thiolate ion is stabilised through the formation of an ion pair with the neighbouring imidazolium group of His 3821. A buried water molecule completes the catalytic triad by forming a hydrogen bond to the N(e2)H of His 3821. This effect of this is to both stabilise the ion pair and also keep the imidazole ring of the His residue in favourable orientation. The oxyanion is stabilised by an oxyanion hole, the amide backbone of Cys 3385 and Gly 3383. Nucleophilic attack of the anionic cysteine S (thiolate ion) on the peptide carbonyl carbon. In this step, a fragment of the substrate is released with an amine terminus, the histidine residue in the protease is restored to its deprotonated form, and a thioester intermediate linking the new carboxy-terminus of the substrate to the cysteine thiol is formed. The thioester bond is subsequently hydrolysed to generate a carboxylic acid moiety on the remaining substrate fragment, whilst regenerating the free enzyme