Human complement proteins D, C2, and B. Active site mapping with peptide thioester substrates.

C.M Kam,B.J Mcrae,J.W Harper,M.A Niemann,J.E Volanakis,J.C Powers

doi:10.1016/s0021-9258(18)61371-7

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.

Highlights

The specificity and reactivity of complement serine (Stroud et al, 1979)
Inthe classical pathway the C3 convertase C4b2a is formed by the sequential cleavage of C4 and C2 by the serine protease In the alternative pathway assembly of the C3 convertase C3bBb is completed by the protein D-catalyzed cleavage of C3bBb or (C3b)-bound protein B
The kinetic parameters katK, m,and kcat/Kmfor the reacenzymes and Clr

Summary

RESULTS

The kinetic parameters katK, m,and kcat/Kmfor the reacenzymes and Clr Numerous thioester substrateswhich werebased on the sequences at the thioester substrates that were not hydrolyzed by these encleavage sites of C3, C5,and B C2 and B to theircatalyticfragments C2a and Bb. The by the NHZ-terminal fragments C2b and Ba. The peptide sensitive synthetic thioester substrates reported here should thioesters which werechosen to map the active site of D, C2, be useful in future active site studiesof complement proteins B, CZa, and Bbhave arginine at PIposition and various amino. Bovine magnifyingglass, Full sizephotocopies are available from the Journal trypsin hydrolyzed all of the thioester substrates rapidly. 'The nomenclature used for the individual amino acid residue (PI, Some of the experimental procedures are described in McRae et Pz,etc.) of a substrate and thesubsites (SI,S2,etc.) of the enzyme is al.

Protein D

NH NH

DISCUSSION

Substrate concentration range kat