title: Expression, characterization and structure determination of an active site mutant (Glu202-Gln) of mini-stromelysin-1.
abstract: Human stromelysin-1 is a member of the matrix metalloproteinase (MMP) family of enzymes. The active site glutamic acid of the MMPs is conserved throughout the family and plays a pivotal role in the catalytic mechanism. The structural and functional consequences of a glutamate to glutamine substitution in the active site of stromelysin-1 were investigated in this study. In contrast to the wild-type enzyme, the glutamine-substituted mutant was not active in a zymogram assay where gelatin was the substrate, was not activated by organomercurials and showed no activity against a peptide substrate. The glutamine-substituted mutant did, however, bind to TIMP-1, the tissue inhibitor of metalloproteinases, after cleavage of the propeptide with trypsin. A second construct containing the glutamine substitution but lacking the propeptide was also inactive in the proteolysis assays and capable of TIMP-1 binding. X-ray structures of the wild-type and mutant proteins complexed with the propeptide-based inhibitor Ro-26-2812 were solved and in both structures the inhibitor binds in an orientation the reverse of that of the propeptide in the pro-form of the enzyme. The inhibitor makes no specific interactions with the active site glutamate and a comparison of the wild-type and mutant structures revealed no major structural changes resulting from the glutamate to glutamine substitution.
authors.affiliation: Department of Biochemistry and Molecular Genetics, Oral Cancer Research Center and Research Center in Oral Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Steele
authors.forename: O
authors.initials: O
authors.lastname: El-Kabbani
authors.forename: P
authors.initials: P
authors.lastname: Dunten
authors.forename: L J
authors.initials: LJ
authors.lastname: Windsor
authors.forename: R U
authors.initials: RU
authors.lastname: Kammlott
authors.forename: R L
authors.initials: RL
authors.lastname: Crowther
authors.forename: C
authors.initials: C
authors.lastname: Michoud
authors.forename: J A
authors.initials: JA
authors.lastname: Engler
authors.forename: J J
authors.initials: JJ
authors.lastname: Birktoft
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D000611:Aminobiphenyl Compounds / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005780:Gelatin / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D019278:Matrix Metalloproteinase 3 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D061965:Matrix Metalloproteinase Inhibitors; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000737:chemistry
chemical_list: D000611:Aminobiphenyl Compounds; D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; C412836:Ro 26-2812; D019715:Tissue Inhibitor of Metalloproteinase-1; D005780:Gelatin; D019278:Matrix Metalloproteinase 3