The Enzymatic Nature of Human C1r̄: A Subcomponent of the First Component of Complement

Abstract

The esterase activity of the Clr̄ subcomponent of the first component of complement has been investigated. Clr̄ was found to hydrolyze two amino acid methyl esters; N-acetyl-L-arginine methyl ester and N-acetyl-glycyl-L-lysine methyl ester, and two amino acid p-nitro-phenyl esters, N-carbobenzyloxy-L-tyrosine-p-nitro-phenyl ester and N^α-carbobenzyloxy-L-lysine-p-nitrophenyl ester. A detailed kinetic analysis of the hydrolysis of N-Z-L-Tyr-ONp by Clr̄ revealed that the enzymatic activity per microgram of protein decreased as the Clr̄ concentration was increased. the loss of activity suggested that above 0.5 µM Clr̄ was undergoing aggregation with a loss of active sites. Similarly, when Clr̄ was titrated with the active site titrant p-nitrophenyl-p-guanidinobenzoate the number of titratable sites per milligram of protein decreased with increasing protein concentration. The hydrolysis of N-Z-L-Tyr-ONp by Clr̄ was inhibited by several synthetic inhibitors including phenylmethanesulfonylfluoride, p-amidinophenylmeth-anesulfonylfluoride, diisopropylfluorophosphate, and p-tosyl-L-lysine-chloromethyl ketone. However, the peptide esterase inhibitors Trasylol, hirudin, leupeptin, and Cl̄ esterase inhibitor had no effect on the esterase activity of Clr̄.