Catalysis of individual steps of protein splicing of the Pyrococcus abyssi PolII intein

Abstract

Protein splicing involves the excision of an intervening polypeptide, the intein, from flanking polypeptides, the exteins, concomitant with the specific ligation of the exteins. The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be over-expressed and purified as an unspliced precursor. This allows for a detailed, in vitro kinetic analysis of the individual steps of protein splicing. We have studied the influence of a conserved intein TXXH motif and of the flanking C-terminal residue of the N-extein on the rate and extent of the first step of protein splicing, an amide-thioester rearrangement of the peptide bond linking the N-extein and intein. We also show that the second step, a trans-thioesterification that results in a branched thioester intermediate, is rapidly reversible with respect to formation of the linear thioester. The third step, Gln cyclization coupled to cleavage of the peptide bond linking the intein and C-extein, is rate determining, and its rate and extent are influenced by two conserved intein His residues. The rate of this step is greatly increased upon substitution of Gln with the highly conserved Asn. Our mechanistic work suggests how the intein can catalyze three distinct chemical reactions in proper sequence, which results in a splicing reaction in place of unproductive side-reactions. This material is based upon work supported by the National Science Foundation (Grants DBI-0320824 and MCB/CAREER-0447647) and by the Research Corporation.