Capturing the active transition state of an LPS-sensitive protease zymogen in autocatalytic activation

Abstract

 Hemolymph coagulation in horseshoe crabs is triggered by the autocatalytic activation of a lipopolysaccharide (LPS) -sensitive serine protease zymogen factor C through its transition state (factor C^＊). However, the existence of factor C^＊ is only speculative, and it remains unknown whether the autocatalytic cleavage of the Phe⁷³⁷-Ile⁷³⁸ bond (the F737 site) of factor C^＊ required for the conversion to an active form α-factor C occurs intramolecularly or intermolecularly. We show that the F737 site of a catalytic Ser⁹⁴¹-deficient mutant of factor C is cleaved by an F737 site-uncleavable mutant in the presence of LPS. These data clearly indicate the existence of factor C^＊ without cleavage of the F737 site. We also found the following facts : (1) the autocatalytic cleavage at the F737 site of factor C^＊ occurs intermolecularly on the LPS surface ; (2) factor C^＊ does not exhibit intrinsic chymotryptic activity against the F737 site during the autocatalytic activation, and (3) LPS is required not only to complete the substrate-binding site and oxyanion hole of factor C^＊ but also to allow the F737 site to be cleaved.