Highly Conserved Histidine Plays a Dual Catalytic Role in Protein Splicing: A pKa Shift Mechanism
| Autor: | Georges Belfort, Zhenming Du, Brian Pereira, Yangzhong Liu, Philip Shemella, Saroj Nayak, Marlene Belfort, Chunyu Wang, Scott A. McCallum |
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| Rok vydání: | 2009 |
| Předmět: |
Models
Molecular Molecular Sequence Data Crystallography X-Ray Biochemistry Molecular mechanics Article Catalysis Inteins Scissile bond Residue (chemistry) Colloid and Surface Chemistry Protein splicing Protein Splicing Histidine Amino Acid Sequence Nuclear Magnetic Resonance Biomolecular biology Chemistry Active site Mycobacterium tuberculosis General Chemistry Hydrogen-Ion Concentration RNA splicing biology.protein Intein Sequence Alignment |
| Zdroj: | Journal of the American Chemical Society. 131:11581-11589 |
| ISSN: | 1520-5126 0002-7863 |
| DOI: | 10.1021/ja904318w |
| Popis: | 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) of3.5 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. |
| Databáze: | OpenAIRE |
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