Specific amino acid substitutions in human collagenase cause decreased autoproteolysis and reveal a requirement for a second zinc atom for catalytic activity.
We have previously reported the crystal structure of truncated human collagenase (domain II) complexed with a low molecular weight inhibitor. Attempts to crystallize 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 1251-->S substitution at the domain II/III junction and demonstrate an increased resistance to proteolysis compared to wild-type collagenase.
Roche Research Centre, Welwyn Garden City, Herts, UK.
D H
DH
Williams
E J
EJ
Murray
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D017364:Collagenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D006706:Homeostasis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D015032:Zinc / Q000494:pharmacology*
D011994:Recombinant Proteins; D017364:Collagenases; D015032:Zinc