Mechanism and Catalytic Site Atlas

Introduction

After synthesis of a nascent protein an internal protein domain (termmed the intein) is excised from the precursor and the two external polypeptides (termed exteins) are ligated together through a peptide bond. Catalysis begins with cleavage of the N-extein-intein peptide bond by nucleophilic attack of the Cys at the intein N terminus on the immediate upstream carbonyl carbon. A tetrahedral intermediate is formed which then collapses with lysis of the C-N bond aided by donation of a proton by His75 and steric strain. The transition state is stabilised in an oxyanion hole formed by Thr72 and Asn74. Transfer of the N-extein to the side chain hydroxyl of the Thr199 at the N terminus of the C-extein follows. This forms a branched intermediate at the Thr199 residue. Cleavage of the intein-C-extein peptide bond occurs by cyclic imide formation of the C-terminal intein Asn198 residue. The tetrahedral intermediate formed by nucleophilic attack of the side chain N on the carbonly group collapses productively due to donation of a proton to the leaving amine group from His197. Thus the intein is lost from the protein with a C terminal succinimide group. The final stage of peptide bond formation may occur without the aid of catalysis. Nucleophilic attack of the newly released free N terminus of C-extein on the ester-linked N- and C-extein junction causes formation of a peptide linkage and reformation of the Thr199 residue.