Cleavage specificity of a proteolytic antibody light chain and effects of the heavy chain variable domain11Edited by A.R.Fersht

The recombinant light chain (L chain) of an antibody raised by immunization with vasoactive intestinal polypeptide (VIP) cleaved this peptide on the C-terminal side of basic residues. The major sites of cleavage in VIP were two adjacent peptide bonds, Lys20Lys21 and Lys21Tyr22. Lower levels of cleavage were evident at Arg14Lys15 and Lys15Gln16. Hydrolysis of radiolabeled VIP by the L chain was inhibited by two serine protease inhibitors, diisopropylfluorophosphate and aprotinin, but not by soybean or lima bean trypsin inhibitors or inhibitors of other classes of proteases. To probe the role of the VHdomain, single chain Fvconstructs composed of the VLdomain of the anti-VIP L chain linked viaa 14-residue peptide to its natural VHdomain partner or an irrelevant anti-lysozyme VHdomain (hybrid Fv) were prepared. The anti-VIP Fvhydrolyzed VIP with Ks21.4-fold lower than the L chain and 250-fold lower than the hybrid Fv, suggesting increased affinity for the substrate ground state due to the anti-VIP VHdomain. The kinetic efficiency (kcat/Ks) of the anti-VIP Fvwas 6.6-fold greater compared to the L chain and 29.4-fold greater compared to the hybrid Fv. Peptide-MCA substrates unrelated in sequence to VIP were hydrolyzed by the anti-VIP Fvand L chain at equivalent rates. These observations lead to a model of catalysis by the anti-VIP Fvin which the essential catalytic residues are located in the VLdomain and additional residues from the VHdomain are involved in high affinity binding of the substrate.