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Journal of Biochemistry Advance Access originally published online on December 15, 2006
Journal of Biochemistry 2007 141(2):251-259; doi:10.1093/jb/mvm026
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© 2006 The Japanese Biochemical Society.

Identification and Characterization of Functional Intermediates of Stem Bromelain During Urea and Guanidine Hydrochloride Unfolding

Basir Ahmad, Tabrez Anwer Shamim, Soghra Khatun Haq and Rizwan Hasan Khan*

Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh–202002, India

*To whom correspondence should be addressed. Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh–202 002, India. Tel: +91-571-2720388, Fax: + 91-571-2721776, E-mail: rizwanhkhan{at}hotmail.com; rizwanhkhan1{at}yahoo.com

Received October 11, 2006; Accepted December 2, 2006


  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.

Key Words: circular dichroism, cysteine protease, functional intermediate state, intrinsic fluorescence, proteolytic activity

Abbreviations: ANS, 1-anilino-8-nephthalenesulphonicacid; CD, circular dichroism; GnHCl, guanidine hydrochloride; PFI, partially folded intermediate; SB, stem bromelain


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