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.
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Research Article|
December 21 2006
Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure
Paul A. O'Farrell;
Paul A. O'Farrell
1
1W.M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, U.S.A.
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Leemor Joshua-Tor
Leemor Joshua-Tor
2
1W.M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, U.S.A.
2To whom correspondence should be addressed (email leemor@cshl.edu).
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Publisher: Portland Press Ltd
Received:
April 29 2006
Revision Received:
September 20 2006
Accepted:
September 28 2006
Accepted Manuscript online:
September 28 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2007
Biochem J (2007) 401 (2): 421–428.
Article history
Received:
April 29 2006
Revision Received:
September 20 2006
Accepted:
September 28 2006
Accepted Manuscript online:
September 28 2006
Citation
Paul A. O'Farrell, Leemor Joshua-Tor; Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure. Biochem J 15 January 2007; 401 (2): 421–428. doi: https://doi.org/10.1042/BJ20060641
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