Cys102 and His398 Are Required for Bleomycin-Inactivating Activity but Not for Hexamer Formation of Yeast Bleomycin Hydrolase
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- 作者：Zhendong Pei and and Saï ; d M. Sebti
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：August 20, 1996
- 年：1996
- 卷：35
- 期：33
- 页码：10751 - 10756
- 全文大小：329K
- 年卷期：v.35,no.33(August 20, 1996)
- ISSN：1520-4995
文摘
The bleomycin-inactivating enzyme, bleomycin hydrolase, isbelieved to be involved in tumorresistance to the anticancer drug bleomycin. This homohexamer isan aminopeptidase that shows homologyto cysteine proteinases around the cysteine and histidine active site.The role that these residues play inhydrolyzing bleomycin and in hexamer oligomerization of bleomycinhydrolase is not known. In thisstudy, the yeast bleomycin hydrolase gene was expressed inEscherichia coli, and site-directed mutagenesiswas employed to precisely investigate the roles of the conservedCys102 and His398 residues in itsstructureand enzymatic activity. Three mutants were created, in whichCys102 was replaced by arginine or serine,and His398 was changed to glycine. The ability ofbleomycin hydrolase to oligomerize was neither affectedby the subtle cysteine/serine mutation nor affected bycysteine/arginine or histidine/glycine mutations.However, the ability of bleomycin hydrolase to hydrolyze andinactivate bleomycin was totally abolishedin all three mutants, suggesting that the cysteine thiol and histidineimidazole are critical for hydrolyzingbleomycin. Furthermore, in contrast to predictions from therecently reported crystal structure of thisenzyme, hexamer formation is not required for the enzymatic activity ofbleomycin hydrolase. Thus,these results demonstrate that Cys102 andHis398 are required for bleomycin hydrolase activity butnothexamer formation, and that both monomer and hexamer are active formsof bleomycin hydrolase.