title: Human complement proteins D, C2, and B. Active site mapping with peptide thioester substrates.
abstract: 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 4,4'-dithiodipyridine at pH 7.5. Each substrate contained a P1 arginine residue, and the effect of various groups and amino acids in the P2, P3, P4, and P5 positions was determined using kcat/Km 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 cleavage sequence of B was hydrolyzed. Overall, D cleaved fewer substrates and was 2-3 orders of magnitude less reactive than C1s 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 Z-Gly-Leu-Ala-Arg-SBzl and Z-Leu-Gly-Leu-Ala-Arg-SBzl, respectively, where Bzl is benzyl. B was the least reactive among these complement enzymes. The best substrate for B was Z-Lys-Arg-SBzl with a kcat/Km value of 1370 M-1 s-1. The catalytic fragment of B, Bb, had higher activity toward these peptide thioester substrates. The best substrate for Bb was Z-Gly-Leu-Ala-Arg-SBzl with a kcat/Km similar to C2a and 10 times higher than the value for B. Both C2a and Bb were considerably more reactive against C3-like than C5-like substrates. Bovine trypsin hydrolyzed thioester substrates with kcat/Km approximately 10(3) 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.
mesh_terms: D000818:Animals; D002417:Cattle; D003166:Complement Activating Enzymes / Q000378:metabolism*; D003175:Complement C2 / Q000302:isolation & purification / Q000378:metabolism*; D011415:Complement Factor B / Q000302:isolation & purification / Q000378:metabolism*; D011416:Complement Factor D / Q000302:isolation & purification / Q000378:metabolism*; D004220:Disulfides; D010450:Endopeptidases / Q000097:blood*; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011725:Pyridines / Q000494:pharmacology*; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism
chemical_list: D003175:Complement C2; D004220:Disulfides; D004792:Enzyme Precursors; D011725:Pyridines; D013439:Sulfhydryl Reagents; C007542:4,4'-dipyridyl disulfide; D003166:Complement Activating Enzymes; D010450:Endopeptidases; D012697:Serine Endopeptidases; D014357:Trypsin; C493488:CFD protein, human; D011416:Complement Factor D; D011415:Complement Factor B