Contribution of the N-terminal region of hirudin to its interaction with thrombin

Abstract

Hirudin is a 65-residue polypeptide that specifically inhibits thrombin by forming a tight, noncovalent complex with the enzyme. The role of the two amino-terminal valine residues and the N-terminal .alpha.-amino group of hirudin in the formation of the complex has been investigated by site-directed mutagenesis and chemical modification. Replacement of the two N-terminal valyl residues of recombinant hirudin by polar amino acids resulted in an increase in the inhibition constant (KI). In contrast, replacement of these residues by hydrophobic amino acids had little effect on the value for KI. These results demonstrated that the hydrophobic nature of the N-terminal residues of hirudin was important for its interaction with thrombin. Addition of a single amino acid to the N-terminus of hirudin resulted in a marked increase in the value of KI. A similar effect was observed when the positive charge of the .alpha.-amino group was removed by acetylation. In contrast, amidination of this group, which preserves the positive charge, resulted in a less pronounced increase in the value of KI. Thus, it appears that a positive charge immediately adjacent to the N-terminal hydrophobic residue is required for optimal binding to thrombin.