O'Farrell, P. A., Joshua-Tor, L. (January 2007) Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure. Biochem J, 401 (2). pp. 421-8. ISSN 0264-6021 (Public Dataset)
Abstract
Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Amino Acid Sequence *Binding Sites Crystallization Crystallography, X-Ray Cysteine Endopeptidases/*chemistry/*genetics Hydrogen Bonding Mutagenesis, Site-Directed Saccharomyces cerevisiae/enzymology Water/chemistry |
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > mutations > mutagenesis Investigative techniques and equipment > x ray crystallography |
| CSHL Authors: | |
| Communities: | CSHL labs > Joshua-Tor lab |
| Depositing User: | Matt Covey |
| Date: | 15 January 2007 |
| Date Deposited: | 17 Jan 2017 19:36 |
| Last Modified: | 05 Sep 2017 18:26 |
| PMCID: | PMC1820812 |
| Related URLs: | |
| Dataset ID: | |
| URI: | https://repository.cshl.edu/id/eprint/34024 |
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