STRUCTURAL BASIS FOR THE BROAD SUBSTRATE-SPECIFICITY OF FIDDLER-CRAB COLLAGENOLYTIC SERINE-PROTEASE-1

Crab collagenolytic serine protease 1 efficiently cleaves peptide bond s directly C-terminal to basic, polar, and hydrophobic amino acids. Th e crystal structure of this enzyme complexed to the protein inhibitor ecotin at 2.5 A resolution reveals a large primary binding pocket punc tuated on one wall by the side chain of aspartate-226. Removal or relo cation of this negatively charged group by site-directed mutagenesis g enerates variant enzymes which retain very high activities toward sele cted substrates. Full retention of activity toward hydrophobic substra tes in collagenase D226G is accompanied by a 10-100-fold reduction in k(cat)/K-m toward basic residues. In contrast, restoration of the nega tive charge in a trypsin-like position in collagenase D226G/G189D rege nerates nearly full activity toward basic substrates while introducing a 5-fold decrease in k(cat)/K-m toward hydrophobic amino acids. These results imply that the collagenase S1 pocket has multiple distinct bi nding sites for different amino acid side chains, a suggestion support ed by molecular modeling studies based on the crystal structure. The e ase of specificity modification in the primary binding site of this se rine protease parallels similar observations with the bacterial enzyme s alpha-lytic protease and subtilisin, and stands in sharp distinction to the extensive mutagenesis required to alter specificity in trypsin .