期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 113, 期 44, 页码 12438-12443出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1612620113
关键词
dual specificity; nitrilase superfamily; N-end rule; Nta1
资金
- National Research Foundation of Korea grants from the Korean government [NRF-2011-0028168]
- Basic Research Laboratory [2015041919]
- Institute for Basic Science Grant [IBS-R023-D1]
- Korean Association of University Woman Fellowship
The first step of the hierarchically organized Arg/N-end rule pathway of protein degradation is deamidation of the N-terminal glutamine and asparagine residues of substrate proteins to glutamate and aspartate, respectively. These reactions are catalyzed by the N-terminal amidase (Nt-amidase) Nta1 in fungi such as Saccharomyces cerevisiae, and by the glutamine-specific Ntaq1 and asparagine-specific Ntan1 Nt-amidases in mammals. To investigate the dual specificity of yeast Nta1 (yNta1)and the importance of second-position residues in Asn/Gln-bearing N-terminal degradation signals (N-degrons), we determined crystal structures of yNta1 in the apo state and in complex with various N-degron peptides. Both an Asn-peptide and a Gln-peptide fit well into the hollow active site pocket of yNta1, with the catalytic triad located deeper inside the active site. Specific hydrogen bonds stabilize interactions between N-degron peptides and hydrophobic peripheral regions of the active site pocket. Key determinants for substrate recognition were identified and thereafter confirmed by using structure-based mutagenesis. We also measured affinities between yNta1 (wild-type and its mutants) and specific peptides, and determined K-M and k(cat) for peptides of each type. Together, these results elucidate, in structural and mechanistic detail, specific deamidation mechanisms in the first step of the N-end rule pathway.
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