Highly Conserved Histidine Plays a Dual Catalytic Role in Protein Splicing: A pK_a Shift Mechanism

Protein splicing is a precise autocatalytic process in which an intein excises itself from a precursor with the concomitant ligation of the flanking sequences. Protein splicing occurs through acid−base catalysis in which the ionization states of active site residues are crucial to the reaction mechanism. In inteins, several conserved histidines have been shown to play important roles in protein splicing, including the most conserved “B-block” histidine. In this study, we have combined NMR pK_a determination with quantum mechanics/molecular mechanics (QM/MM) modeling to study engineered inteins from Mycobacterium tuberculosis (Mtu) RecA intein. We demonstrate a dramatic pK_a shift for the invariant B-block histidine, the most conserved residue among inteins. The B-block histidine has a pK_a of 7.3 ± 0.6 in a precursor and a pK_a of <3.5 in a spliced intein. The pK_a values and QM/MM data suggest that the B-block histidine has a dual role in the acid−base catalysis of protein splicing. This histidine likely acts as a general base to initiate splicing with an acyl shift and then as a general acid to cause the breakdown of the scissile bond at the N-terminal splicing junction. The proposed pK_a shift mechanism accounts for the biochemical data supporting the essential role for the B-block histidine and for the near absolute sequence conservation of this residue.