title: Importance of the prime subsites of the C1s protease of the classical complement pathway for recognition of substrates.
abstract: The classical complement pathway, which plays a vital role in preventing infection, is initiated by the action of the serine proteases C1r and C1s. We have examined the hydrolysis of substrates representing cleavage sequences in the physiological substrates for C1s, C2 and C4. These studies showed that the P(1)'-P(4)' substrate residues of C2 and C4 conferred greater affinity of substrate for enzyme and also induced a sigmoidal dependence of enzyme velocity on substrate concentration. This indicates that the substrate gave rise to homotropic positive cooperative behavior in the enzyme. When C1s was in complex with C1q and C1r, as would occur under physiological conditions, the same behavior was observed, indicating that this mechanism is relevant in the complement pathway in vivo. We further investigated the requirements of C1s for prime side amino acids by examining a substrate library in which each of the P(1)'-P(4)' positions had been substituted by different classes of amino acids. This revealed that the P(1)' position was a major determinant of the selectivity of the enzyme, while certain substitutions at the P(1)'-P(4)' positions abolished the allosteric behavior, indicating that contact residues at these positions in the C1s enzyme must mediate the cooperativity. The studies reported here highlight the importance of prime subsites in C1s for interaction with its cognate substrates in the complement pathway and therefore yield greater understanding of the mechanism of interaction between this vital protease and its physiological substrates.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Grace
authors.initials: G
authors.lastname: O'Brien
authors.forename: Noelene S
authors.initials: NS
authors.lastname: Quinsey
authors.forename: James C
authors.initials: JC
authors.lastname: Whisstock
authors.forename: Robert N
authors.initials: RN
authors.lastname: Pike
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D003172:Complement C1 / Q000737:chemistry / Q000378:metabolism; D003173:Complement C1s / Q000737:chemistry* / Q000378:metabolism; D003175:Complement C2 / Q000737:chemistry / Q000378:metabolism; D003181:Complement C4 / Q000737:chemistry / Q000378:metabolism; D003171:Complement Pathway, Classical; D003374:Coumarins / Q000138:chemical synthesis / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D019151:Peptide Library; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D003172:Complement C1; D003175:Complement C2; D003181:Complement C4; D003374:Coumarins; D009842:Oligopeptides; D019151:Peptide Library; D012697:Serine Endopeptidases; D003173:Complement C1s