Human Complement Proteins D , C 2 , and B

The specificity and reactivity of complement serine proteases D, B, Bb, C2, and C2a were determined using a series of peptide thioester substrates. The rates of thioester hydrolysis were measured using assay mixtures containing the thiol reagent I,lt’-dithiodipyridine at pH 7.5. Each substrate contained a PI arginine residue, and the effect of various groups and amino acids in the Pz, P3, P4, and P6 positions was determined using k,,JK, values to compare reactivities. Among peptide thioesters corresponding to the activation site sequence in B, dipeptide thioesters containing a P2 lysine residue were the best substrates for D. Extending the chain to include a P3 or P4 amino acid resulted in loss of activity, and neither the tripeptide nor the tetrapeptide containing the cl avage sequence of B was hydrolyzed. Overall, D cleaved fewer substrates was 2-3 orders of magnitude less reactive than Cls against some thioester substrates. C2 and fragment C2a had comparable reactivities and hydrolyzed peptides containing Leu-Ala-Arg and Leu-Gly-Arg, which have the same sequence as the cleavage sites of C3 and C5, respectively. The best substrates for C2 and C2a were 2-Gly-Leu-Ala-Arg-SBzl and Z-Leu-Gly-LeuAla-Arg-SBzl, respectively, where Bzl is benzyl. B was the least reactive among these complement enzymes. The best substrate for B was 2-Lys-Arg-SBzl with a k,,,/K, value of 1370 M” s-*. The catalytic fragment of B, Bb, had higher activity toward these peptide thioester substrates. The best substrate for Bb was ZGly-Leu-Ala-Arg-SBzl with a k&K, similar to C2a and 10 times higher than the value for B. Both C2a and Bb were considerably more reactive against C3like than C5-like substrates. Bovine trypsin hydrolyzed thioester substrates with k&K, approximately lo3 higher than the complement enzymes. These thioester substrates for D, B, and C2 should be quite useful in kinetic and active site studies of the purified enzymes.