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Specific amino acid substitutions in human collagenase cause decreased autoproteolysis and reveal a requirement for a second zinc atom for catalytic activity
Author(s) -
Williams D.H.,
Murray E.J.
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)01136-2
Subject(s) - collagenase , chemistry , proteolysis , zinc , cleave , amino acid , enzyme , substrate (aquarium) , active site , stereochemistry , biophysics , biochemistry , biology , ecology , organic chemistry
We have previously reported the crystal structure of truncated human collagenase (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 junction at high protein concentrations. To overcome this problem, we have generated an inactive enzyme via a H149 → L,D151 → N double substitution 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 junction and demonstrate an increased resistance to proteolysis compared to wild‐type collagenase.