Enzyme Kinetic Studies of Subcomponent C1r of the First Component of Complement with Peptide Thioester Substrates and Benzamidine inhibitors
Clr is the active serine protease subcomponent of the first component of complement, Cl. Enzyme kinetics were used to study Clr with two synthetic peptide thioesters, one a peptide of lysine, the other a dipeptide of glycine and arginine. The kinetics of the reactions of Clr and the two substrates was measured photometrically by the use of a chromagen. Clr was further characterized by measuring the inhibition constants of benzamidine and its derivatives with Clr and each peptide thioester. The activation energy for Clr with both substrates was determined. Our results demonstrate that Clr was more specific for Z-GLY-ARG-SBzL which was expected since arginine is the likely primary binding site of Cls, Clr's only known in vivo substrate. The inhibitors, which mimic lysine and arginine side chains, were found to inhibit Clr competitively. Use of the inhibitors showed those inhibitors with longer side chains inhibited Clr interaction with either substrate to a greater degree. ClF with either substrate gave essentially the same energy of activation.