SPECIFIC AMINO-ACID SUBSTITUTIONS IN HUMAN COLLAGENASE CAUSE DECREASED AUTOPROTEOLYSIS AND REVEAL A REQUIREMENT FOR A 2ND ZINC ATOM FOR CATALYTIC ACTIVITY

We have previously reported the crystal structure of truncated human c ollagenase (domain II) complexed with a low molecular weight inhibitor . Attempts to crystallise full-length active collagenase (i.e. domain II + III) have been hindered by autoproteolysis at the domain II/III j unction at high protein concentrations. To overcome this problem, we h ave generated an inactive enzyme via a H149 --> L,D151 --> N double su bstitution which displaces the non-catalytic zinc atom, and shown that the altered collagenase is unable to cleave a synthetic substrate. We have also generated an I251 --> S substitution at the domain II/III j unction and demonstrate an increased resistance to proteolysis compare d to wild-type collagenase.