Abstract:
:Haemadin is a 57-amino acid thrombin inhibitor from the land-living leech Haemadipsa sylvestris, whose structure has recently been determined in complex with human alpha-thrombin. Here we communicate the effect of ionic strength on the kinetics of the inhibition of human alpha-thrombin by haemadin, by using thrombin mutants modified in exosite II. Data analysis has allowed both the ionic and nonionic binding contributions to be ascertained, with the nonionic component being virtually the same for all of the thrombins that have been examined, while the ionic binding energy contributions varied from molecule to molecule. In the case of the native human alpha-thrombin-haemadin complex, ionic interactions contribute -17 kJ/mol to the Gibbs free energy of binding, this being the equivalent of up to six salt bridges. These salt bridges make up 20% of the total binding energy at zero ionic strength, and this has been attributed to the C-terminal tail alone. In addition, the contributions of the N-terminal and C-terminal regions of haemadin to its tight binding have been ascertained by using derivatives of both haemadin and thrombin. Limited proteolysis using formic acid produced haemadin cleaved between residues 40 and 41, removing the majority of the C-terminal tail. This truncated haemadin displayed a 20000-fold reduced affinity for thrombin, and was no longer a tight binding inhibitor. A form of thrombin in which the active site serine has been blocked by diisopropyl fluorophosphate binds to haemadin, but with a 72000-fold reduced affinity, indicating that the N-terminus is more important than the C-terminus for strong binding.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Richardson JL,Fuentes-Prior P,Sadler JE,Huber R,Bode Wdoi
10.1021/bi011605qsubject
Has Abstractpub_date
2002-02-26 00:00:00pages
2535-42issue
8eissn
0006-2960issn
1520-4995pii
bi011605qjournal_volume
41pub_type
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