Highly Conserved Histidine Plays a Dual Catalytic Role in Protein Splicing: A p<i>K</i><sub>a</sub> Shift Mechanism Zhenming Du Philip T. Shemella Yangzhong Liu Scott A. McCallum Brian Pereira Saroj K. Nayak Georges Belfort Marlene Belfort Chunyu Wang 10.1021/ja904318w.s001 https://acs.figshare.com/articles/journal_contribution/Highly_Conserved_Histidine_Plays_a_Dual_Catalytic_Role_in_Protein_Splicing_A_p_i_K_i_sub_a_sub_Shift_Mechanism/2834590 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 p<i>K</i><sub>a</sub> determination with quantum mechanics/molecular mechanics (QM/MM) modeling to study engineered inteins from <i>Mycobacterium tuberculosis</i> (<i>Mtu</i>) RecA intein. We demonstrate a dramatic p<i>K</i><sub>a</sub> shift for the invariant B-block histidine, the most conserved residue among inteins. The B-block histidine has a p<i>K</i><sub>a</sub> of 7.3 ± 0.6 in a precursor and a p<i>K</i><sub>a</sub> of <3.5 in a spliced intein. The p<i>K</i><sub>a</sub> 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 p<i>K</i><sub>a</sub> 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. 2009-08-19 00:00:00 RecA intein intein excises histidine acid NMR pKa determination Dual Catalytic Role scissile bond ionization states reaction mechanism sequence conservation Mycobacterium tuberculosis autocatalytic process site residues Protein Splicing role pKa Shift MechanismProtein pKa shift mechanism accounts pKa values acyl shift Conserved Histidine Plays pKa shift QM