Identification of amino acid residues in bone morphogenetic protein-1 important for procollagen C-proteinase activity

Bone morphogenetic protein (BMP)-1, which belongs to the tolloid subgroup o f astacin-like zinc metalloproteinases, cleaves the C-propeptides of procol lagen at the physiologic site and is, therefore, a procollagen C-proteinase (PCP), Cleavage occurs between a specific alanine or glycine residue (depe nding on the procollagen chain) and an invariant aspartic acid residue in e ach of the three chains of procollagen. To learn more about how BMP-1 exhib its PCP activity we mapped the primary structure of BMP-I onto the x-ray cr ystal structure of astacin and identified residues in the metalloproteinase domain of BMP-1 for subsequent site-directed mutagenesis studies. Recombin ant wild-type and mutant BMP-1 were expressed in COS-7 cells and assayed fo r PCP activity using type I procollagen as the substrate. We showed that su bstitution of alanine for Glu(94), which occurs in the HEXXH zinc-binding m otif of BMP-1, abolishes PCP activity. Furthermore, mutation of residues Ly s(87) and Lys(176), Which are located in the S1 ' pocket of the enzyme and are therefore adjacent to the P1 ' residue in the substrate, reduced the pr oteolytic activity of BMP-1 by similar to 50%. A surprising observation was that mutation of Cys(66) reduced the activity to 20%, suggesting that this residue is crucial for activity, Further experiments showed that Cys(66) a nd Cys(63) which are located in the tolloid-specific sequence Cys(63)-Gly(6 4)-Cys(65)-Cys(66) in the active site, most likely form a disulfide bridge.