Structural basis for broad substrate specificity of earthworm fibrinolytic enzyme component A

Abstract

Earthworm fibrinolytic enzyme component A (EFE-a) possesses an S1 1 Nomenclature for the substrate amino acid residues is P n, … , P2, P1, P1′, P2′, … , P n′, where P1–P1′ denotes the hydrolyzed bond. S n, … , S2, S1, S1′, S2′, … , S n′ denotes the corresponding enzyme binding sites. 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 Chymotrypsinogen numbering is used throughout. 2 might endow EFE-a with this specificity. Based on the native crystal structure at a resolution of 2.3 Å, we improved the native crystal structure to 1.8 Å and determined its complex structure with the inhibitor Meo-Suc-Ala-Ala-Pro-Val-CMK at a resolution of 1.9 Å. 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.