Earthworm fibrinolytic enzyme component A (EFE-a) possesses an S1(1) pocket, which is typical for an elastase-like enzyme, but it can still hydrolyze varieties of substrates, and it exhibits wide substrate specificity. Former structure studies suggested that the four-residue insertion after Val(217) (2) might endow EFE-a with this specificity. Based on the native crystal structure at a resolution of 2.3 Angstrom, we improved the native crystal structure to 1.8 Angstrom and determined its complex structure with the inhibitor Meo-Suc-Ala-Ala-Pro-Val-CMK at a resolution of 1.9 Angstrom. The final structures show that: (1) EFE-a possesses multisubstrate-binding sites interacting with the substrates; (2) significant conformation adjustment takes place at two loops binding to the N-terminal of the substrates, which may enhance the, interaction between the enzyme and the substrates. These characteristics make the substrate-specificity of EFE-a less dependent on the property of its S1-pocket and may endow the enzyme with the ability to hydrolyze chymotrypsin-specific substrates and even trypsin-specific substrates. (C) 2004 Elsevier Inc. All rights reserved.