Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin.
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 approximately 2 kcal/mol (1 cal identical with 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.
Department of Pharmaceutical Sciences and CRIBI Biotechnology Center, University of Padua, via F. Marzolo 5, I-35131 Padua, Italy.
Vincenzo
V
De Filippis
Elisa
E
De Dea
Filippo
F
Lucatello
Roberta
R
Frasson
D019943:Amino Acid Substitution; D004795:Enzyme Stability / Q000187:drug effects; D006629:Hirudins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D012964:Sodium / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D012996:Solutions / Q000737:chemistry; D013379:Substrate Specificity / Q000187:drug effects; D013816:Thermodynamics; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism*
D006629:Hirudins; D012996:Solutions; D012964:Sodium; D013917:Thrombin