Modulation of thrombin-hirudin interaction by specific ion effects.
Kinetic studies of the inhibition of thrombin amidase activity by recombinant hirudin have been conducted as a function of salt concentration in the range 0.05 to 1 M, using NaCl, KCl, NaBr and KBr. At the same ionic strength, the value of KI for thrombin-hirudin interaction is found to be different with different salts. The slope d ln KI/d ln a+/-, where a+/- is the mean ion activity, is constant in the range 0.05 to 0.5 M, is sensitive to the particular salt present in solution and is equal to 1.07 +/- 0.09 (NaCl), 0.92 +/- 0.10 (KCl), 1.37 +/- 0.10 (NaBr) and 0.56 +/- 0.10 (KBr). These results indicate that specific ion effects are involved in the modulation of thrombin-hirudin interaction in the form of ion release, as recently found in the case of thrombin interaction with its natural substrate fibrinogen. The linkage hierarchy for ion release found in the case of thrombin-fibrinogen interaction also applies in the case of thrombin-hirudin interaction, with the number of released ions decreasing in the order NaBr greater than NaCl greater than KCl greater than KBr. It is proposed that the process of bridge-binding to the fibrinogen recognition site and the catalytic pocket of the enzyme, as seen in the case of fibrinogen and hirudin, is linked to ion release and controlled by modulation of the association rate constant.
De Cristofaro
R
R
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110.
Fenton
J W
JW
Di Cera
E
E
D001965:Bromides / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006629:Hirudins / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*
D001965:Bromides; D002712:Chlorides; D006629:Hirudins; D046911:Macromolecular Substances; D005340:Fibrinogen; D012964:Sodium; D013917:Thrombin; D011188:Potassium