Hydrolysis of a Synthetic Amide Substrate by Human C1 Esterase (C1s̄)

Abstract

Abstract C1 esterase (C1s̄), the enzymatically active form of the C1s subunit of the first component of complement, activates the classical complement pathway by limited proteolysis of complement components C2 and C4. In searching for chromogenic amide substrates which would reflect the physiologic function of C1s̄, it was found that preparations of C1s̄ slowly hydrolyzed N-benzoyl-L-phenylalanyl-L-valyl-L-arginine p-nitroanilide (BzPheValArgNA). Chromatographic, electrophoretic, and inhibition studies confirmed that the BzPheValArgNA-hydrolyzing activity of these preparations was attributable to C1s̄. Although C1s̄ is known to cleave a number of N-acylated α-amino acid esters, no synthetic amide substrate has been reported previously for this enzyme. Kinetic analysis of the hydrolysis of BzPheValArgNA by C1s̄ yielded an apparent Km of 0.36 mM and a Kcat of 5.7 min-1. Enzymatic activity toward BzPheValArgNA appeared to be controlled by two ionizable groups with pKs of approximately 6.5 and 8.9. In view of the sequence homology between C1 esterase and the pancreatic serine proteases, it is suggested that these groups correspond respectively to the β-carboxyl group of aspartic acid 102 and the α-amino group of isoleucine 16 in α-chymotrypsin. Although C1s̄ hydrolyzed BzPheValArgNA, it did not cleave N-benzoyl-DL-arginine p-nitroanilide. This finding suggests that substrate binding at the subsites occupied by the valyl and/or phenylalanyl residues is critical to the amidase activity of C1s̄.