Structural Stability and Functional Analysis of L-Asparaginase from Pyrococcus furiosus

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S. Bansal¹, D. Gnaneswari¹, P. Mishra¹, and B. Kundu^1,2*

¹Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India

²School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India; fax: 91(11)2658-2282; E-mail: kundudr@yahoo.com; bxk@dbeb.iitd.ac.in; bkundu@bioschool.iitd.ac.in

^* To whom correspondence should be addressed.

Received May 29, 2009; Revision received July 17, 2009

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We report studies on an L-asparaginase from Pyrococcus furiosus, cloned and expressed in Escherichia coli and purified to homogeneity. Protein stability and enzyme kinetic parameters were determined. The enzyme was found to be thermostable, natively dimeric, and glutaminase-free, with optimum activity at pH 9.0. It showed a K_m of 12 mM and a substrate inhibition profile above 20 mM L-asparagine. Urea could not induce unfolding and enzyme inactivation; however, with guanidine hydrochloride (GdnCl) a two-state unfolding pattern was observed. Reduced activity and an altered near-UV-CD signal for protein at low GdnCl concentration (1 M) suggested tertiary structural changes at the enzyme active site. A homology three-dimensional model was developed and the structural information was combined with activity and stability data to give functional clues about the asparaginase.

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KEY WORDS: Pyrococcus furiosus, L-asparaginase, hyperthermophile, chemical denaturation, homology model, stability, enzyme kinetics

DOI: 10.1134/S0006297910030144

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