Mutations in the catalytic domain of prohormone convertase 2 result in decreased binding to 7B2 and loss of inhibition with 7B2 C-terminal peptide

Prohormone convertases 1 (PC1) and 2 (PC2) are members of a family of subti lisin-like proprotein convertases responsible for proteolytic maturation of a number of different prohormones and proneuropeptides. Although sharing m ore than 50% homology in their catalytic domains, PC1 and PC2 exhibit diffe rences in substrate specificity and susceptibility to inhibitors. In additi on to these differences, PC2, unlike PC1 and other members of the family, s pecifically binds the neuroendocrine protein 7B2. In order to identify dete rminants responsible for the specific properties of the PC2 catalytic domai n, we compared its primary sequence with that of other PCs. This allowed us to distinguish a PCS-specific sequence at positions 242-248. We constructe d two PC2 mutants in which residues 242 and 243 and residues 242-248 were r eplaced with the corresponding residues of PC1. Studies of in vivo cleavage of proenkephalin, in vivo production of alpha-MSH from proopiomelanocortin , and in vitro cleavage of a PCB-specific artificial substrate by mutant PC 2s did not reveal profound alterations. On the other hand, both mutant pro- PCBs exhibited a considerably reduced ability to bind to 21-kDa 7B2. In add ition, inhibition of mutant PC2-(242-248) by the potent natural inhibitor 7 B2 CT peptide was almost completely abolished. Taken together, our results show that residues 242-248 do not play a significant role in defining the s ubstrate specificity of PC2 but do contribute greatly to binding 7B2 and ar e critical for inhibition with the 7B2 CT peptide.