THE SUBSTRATE-SPECIFICITY OF UCA PUGILATOR COLLAGENOLYTIC SERINE-PROTEASE-1 CORRELATES WITH THE BOVINE TYPE-I COLLAGEN CLEAVAGE SITES

Affinity-based purification and characterization of the collagenolytic serine protease 1 from Uca pugilator (fiddler crab) hepatopancreas sh ows that the enzyme cleaves the native bovine alpha 1(I) collagen chai n carboxyl-terminal to Gln and Arg residues adjacent to the metallocol lagenase site. Cleavage carboxyl-terminal to Leu residues is observed in the alpha 2(I) chain and at a secondary site in alpha 1(I). These s ites correlate with the prefer ences observed toward p-nitroanilide su bstrates varying at the P1 position, for which the specificity (k(cat) /K-m) is Arg > Leu, Phe, Lys > Gin > Ala. Furthermore, collagen cleava ge after Gln was found exclusively between two Gln-Arg bonds. The P'1- P'3 specificity of collagenase, as determined by nucleophile acyl tran sfer, indicated a strong preference for Arg in the P'1 position. Crab collagenase cleaves peptide bonds adjacent to Leu and Gln at the P1 po sition more efficiently than trypsin, chymotrypsin, or elastase. Moreo ver, the efficiency of collagenase toward P1-Arg substrates is equival ent to that of trypsin. Crystals of crab collagenase have been grown c omplexed with the protein inhibitor ecotin. These crystals diffract to better than 2.8 Angstrom resolution and belong to the space group P3( 2)21 with unit cell dimensions of a = b = 89.0 Angstrom, c = 291.7 Ang strom.