Pei Z, Sebti S M
Department of Pharmacology, School of Medicine, University of Pittsburgh, Pennsylvania 15261, USA.
Biochemistry. 1996 Aug 20;35(33):10751-6. doi: 10.1021/bi9605447.
The bleomycin-inactivating enzyme, bleomycin hydrolase, is believed to be involved in tumor resistance to the anticancer drug bleomycin. This homohexamer is an aminopeptidase that shows homology to cysteine proteinases around the cysteine and histidine active site. The role that these residues play in hydrolyzing bleomycin and in hexamer oligomerization of bleomycin hydrolase is not known. In this study, the yeast bleomycin hydrolase gene was expressed in Escherichia coli, and site-directed mutagenesis was employed to precisely investigate the roles of the conserved Cys102 and His398 residues in its structure and enzymatic activity. Three mutants were created, in which Cys102 was replaced by arginine or serine, and His398 was changed to glycine. The ability of bleomycin hydrolase to oligomerize was neither affected by the subtle cysteine/serine mutation nor affected by cysteine/arginine or histidine/glycine mutations. However, the ability of bleomycin hydrolase to hydrolyze and inactivate bleomycin was totally abolished in all three mutants, suggesting that the cysteine thiol and histidine imidazole are critical for hydrolyzing bleomycin. Furthermore, in contrast to predictions from the recently reported crystal structure of this enzyme, hexamer formation is not required for the enzymatic activity of bleomycin hydrolase. Thus, these results demonstrate that Cys102 and His398 are required for bleomycin hydrolase activity but not hexamer formation, and that both monomer and hexamer are active forms of bleomycin hydrolase.
