期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 131, 期 32, 页码 11581-11589出版社
AMER CHEMICAL SOC
DOI: 10.1021/ja904318w
关键词
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资金
- National Institute of Health [R01 GM81408, R01GM44844]
- National Science Foundation [CTS03-04055-NIRT]
- Computational Center for Nanotechnology Innovations (CCNI)
- Interconnect Focus Center
- Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China [707036]
- NIH Biomolecular Science and Engineering training [GM067545]
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.
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