NDLI: Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin

Content Provider	PubMed Central
Author	Filippis, Vincenzo De Dea, Elisa De Filippo, Lucatello Frasson, Roberta
Copyright Year	2005
Abstract	In the present work, the effect of Na+ binding on the conformational, stability and molecular recognition properties of thrombin was investigated. The binding of Na+ reduces the CD signal in the far-UV region, while increasing the intensity of the near-UV CD and fluorescence spectra. These spectroscopic changes have been assigned to perturbations in the environment of aromatic residues at the level of the S2 and S3 sites, as a result of global rigidification of the thrombin molecule. Indeed, the Na+-bound form is more stable to urea denaturation than the Na+-free form by ∼2 kcal/mol (1 cal≡4.184 J). Notably, the effects of cation binding on thrombin conformation and stability are specific to Na+ and parallel the affinity order of univalent cations for the enzyme. The Na+-bound form is even more resistant to limited proteolysis by subtilisin, at the level of the 148-loop, which is suggestive of the more rigid conformation this segment assumes in the ‘fast’ form. Finally, we have used hirudin fragment 1–47 as a molecular probe of the conformation of thrombin recognition sites in the fast and ‘slow’ form. From the effects of amino acid substitutions on the affinity of fragment 1–47 for the enzyme allosteric forms, we concluded that the specificity sites of thrombin in the Na+-bound form are in a more open and permissible conformation, compared with the more closed structure they assume in the slow form. Taken together, our results indicate that the binding of Na+ to thrombin serves to stabilize the enzyme into a more open and rigid conformation.
Related Links	http://dx.doi.org/10.1042/BJ20050252
Starting Page	485
File Format	PDF
ISSN	14708728
e-ISSN	14708728
Journal	Biochemical Journal
Issue Number	Pt 2
Volume Number	390
Language	English
Publisher	Portland Press Ltd.
Publisher Date	2005-09-01
Access Restriction	Open
Rights Holder	Portland Press Ltd.
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology Biochemistry

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