Cleavage specificity of a proteolytic antibody light chain and effects of the heavy chain variable domain

Abstract 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, Lys20 Lys21 and Lys21 Tyr22. Lower levels of cleavage were evident at Arg14 Lys15 and Lys15 Gln16. 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 V H domain, single chain F v constructs composed of the V L domain of the anti-VIP L chain linked via a 14-residue peptide to its natural V H domain partner or an irrelevant anti-lysozyme V H domain (hybrid F v ) were prepared. The anti-VIP F v hydrolyzed VIP with K s 21.4-fold lower than the L chain and 250-fold lower than the hybrid F v , suggesting increased affinity for the substrate ground state due to the anti-VIP V H domain. The kinetic efficiency ( k cat / K s ) of the anti-VIP F v was 6.6-fold greater compared to the L chain and 29.4-fold greater compared to the hybrid F v . Peptide-MCA substrates unrelated in sequence to VIP were hydrolyzed by the anti-VIP F v and L chain at equivalent rates. These observations lead to a model of catalysis by the anti-VIP F v in which the essential catalytic residues are located in the V L domain and additional residues from the V H domain are involved in high affinity binding of the substrate.