Mapping the substrate binding site of human C1r and C1s with peptide thioesters. Development of new sensitive substrates

• Published in: The Journal of biological chemistry Vol. 256; no. 23; pp. 12362 - 12366
• Main Authors: McRae, B J, Lin, T Y, Powers, J C
• Format: Journal Article
• Language: English
• Published: United States 10.12.1981
• Subjects: Binding Sites; Complement Activating Enzymes - metabolism; Complement C1r; Complement C1s; Enzyme Precursors - metabolism; Humans; Kinetics; man; Peptides - chemical synthesis; Protein Binding; Substrate Specificity; Sulfhydryl Compounds - pharmacology
• ISSN: 0021-9258
• DOI: 10.1016/S0021-9258(18)43280-2

The subsite specificities of subcomponents C1r and C1s of human complement C1 were mapped with amino acid and peptide thioester substrates. 4,4'-dithiodipyridine was used to measure the rates of thiol release at pH 7.5 as the various substrates were hydrolyzed by C1r and C1s. Each substrate had a P sub(1) Arg residue, and the S sub(2) and S sub(1) subsites of the enzymes were studied by varying the P sub(2) amino acid residue and the thiol leaving group (P' sub(1)). The K sub(cat)/K sub(m) values were used to compare the relative reactivities of these substrates. Peptide thioesters with sequences identical to the COOH-terminal sequences at the activation sites of C3, C4, and C5 were also investigated. Human C1r exhibited lower reactivity and a higher substrate specificity than C1s. It preferred a Gly in the P sub(2) position. The longer peptide thioesters containing a P sub(2) Gly were less reactive than the dipeptide Z-Gly-Arg-SBzl, as were the P sub(2) Ala and Gln thioesters.