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