title: Identification and characterization of functional intermediates of stem bromelain during urea and guanidine hydrochloride unfolding.
abstract: By comparing changes in enzyme activity with changes in spectral features for stem bromelain (EC.3.4.22.32) in the absence and presence of urea, Guanidine hydrochloride and ethanol; four intermediate states could be identified: two activity-enhanced state obtained in the presence of 5 M urea and 2 M GnHCl, termed X and X', respectively, and a third, similarly active state closely resembling the native protein in the presence of 8-9 M urea, termed Y. The enhanced activity of these states is due to local conformational changes accompanied by increased dynamics in the active site. Further, the enzyme does not lose its activity after substantial tertiary structure changes in 8-9 M urea (Y state), suggesting that active site containing domain is more resistant to chemical denaturation than the other structural domain. This makes stem bromelain and in general cysteine proteases an exception to the hypothesis that active site is the most labile part of enzyme.
authors.affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India.
authors.forename: Basir
authors.initials: B
authors.lastname: Ahmad
authors.forename: Tabrez Anwer
authors.initials: TA
authors.lastname: Shamim
authors.forename: Soghra Khatun
authors.initials: SK
authors.lastname: Haq
authors.forename: Rizwan Hasan
authors.initials: RH
authors.lastname: Khan
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001963:Bromelains / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D000431:Ethanol / Q000494:pharmacology; D005453:Fluorescence; D019791:Guanidine; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D012997:Solvents / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014508:Urea
chemical_list: D000817:Anilino Naphthalenesulfonates; D012997:Solvents; D000431:Ethanol; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D001963:Bromelains; C091760:stem bromelain; D019791:Guanidine