Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure

O'Farrell, P. A., Joshua-Tor, L. (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

URL: https://www.ncbi.nlm.nih.gov/pubmed/17007609
DOI: 10.1042/bj20060641

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 Deposited: 17 Jan 2017 19:36
Last Modified: 05 Sep 2017 18:26
PMCID: PMC1820812
Related URLs:
Dataset ID:
URI: http://repository.cshl.edu/id/eprint/34024

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