Essential binding and functional domains of human bleomycin hydrolase

被引:30
作者
Koldamova, RP
Lefterov, IM
Gadjeva, VG
Lazo, JS
机构
[1] Univ Pittsburgh, Sch Med, Dept Pharmacol, Pittsburgh, PA 15261 USA
[2] Univ Pittsburgh, Inst Canc, Pittsburgh, PA 15261 USA
关键词
D O I
10.1021/bi9722204
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Bleomycin hydrolase (BM) is unusual among cysteine proteinases because it appears to form multihomomeric structures, inactivates the antitumor glycopeptide bleomycin. and contains a unique C-terminal amino acid sequence. We now demonstrate intrinsic endopeptidase activity associated with human BH (hBH) using artificial substrates and intracellular dimerization of hBH using a yeast two-hybrid assay, To determine domains important for homomeric interactions and catalysis, we constructed N- and C-terminal deletion mutants and identified an N-terminal region (hBH(1-82)) that interacted with two nonoverlaping hBH domains: one near the N-terminus (hBH(14-103);) and another neighboring the C-terminus (hBH(358-455)) In vitro hBH aggregated with a molecular mass of 235 kD corresponding to a homotetramer and the C-terminus was critical for this oligomerization since no tetramers were found when the last 40 amino acids were deleted, The penultimate 8 amino acids, which constitute a unique and highly conserved bleomycin hydrolase-like domain (BHYD), were essential for BH and aminopeptidase activity but not for endopeptidase activity or oligomer formation. Thus, the C-terminus of hBH has two independent I roles controlling both the catalytic activity and oligomerization of hBH.
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页码:2282 / 2290
页数:9
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