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Essential binding and functional domains of human bleomycin hydrolase
R P Koldamova1, I M Lefterov, V G Gadjeva
1Department of Pharmacology, University of Pittsburgh, School of Medicine, and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15261, USA.
Biochemistry
|March 28, 1998
Summary
Human bleomycin hydrolase (hBH) exhibits endopeptidase activity and forms homotetramers. Its C-terminus regulates both catalytic function and oligomerization, with a unique domain essential for specific enzyme activities.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Bleomycin hydrolase (BH) is a cysteine proteinase with unusual properties, including multihomomeric structure formation and inactivation of the antitumor agent bleomycin.
- BH possesses a unique C-terminal amino acid sequence.
- Understanding the structural and functional domains of BH is crucial for its biochemical characterization.
Purpose of the Study:
- To investigate the intrinsic endopeptidase activity of human BH (hBH).
- To explore the mechanisms of intracellular dimerization and homomeric interactions in hBH.
- To identify specific domains responsible for hBH's catalytic activity and oligomerization.
Main Methods:
- Utilized artificial substrates to demonstrate intrinsic endopeptidase activity of hBH.
- Employed a yeast two-hybrid assay to confirm intracellular dimerization of hBH.
- Constructed N- and C-terminal deletion mutants to map functional domains.
Main Results:
- hBH was shown to possess intrinsic endopeptidase activity and form homotetramers in vitro (235 kD).
- An N-terminal region (hBH1-82) interacted with two distinct hBH domains (hBH14-103 and hBH358-455).
- The C-terminus, particularly the penultimate 8 amino acids (BHYD), was critical for oligomerization and enzyme activity, but not endopeptidase function.
Conclusions:
- The C-terminus of hBH plays dual roles, regulating both catalytic activity and oligomerization.
- The unique bleomycin hydrolase-like domain (BHYD) is essential for BH and aminopeptidase activity.
- hBH's ability to form homotetramers is dependent on its C-terminal region.