title: Zinc ligands in an astacin family metalloprotease meprin A.
abstract: A conserved tyrosine residue in the 'astacin family' of metalloproteases is one of five ligands proposed to coordinate zinc at the active site. Site-directed mutagenesis of the conserved Tyr (Y226) of recombinant mouse meprin alpha was used to test the hypothesis that this residue is essential for zinc binding and enzymatic activity. In addition, another proposed zinc binding ligand, H167, in the conserved (HEXXH) zinc binding motif of the meprin alpha protease domain was replaced by an alanine residue. Both mutants were expressed and secreted with the same subunit mass as wild type (90 kDa). The Y226F mutant retained the capacity to oligomerize to higher covalently and noncovalently-linked oligomers as the wild type, whereas H167A was predominantly a monomer. The kcat/Km for Y226F against a fluorgenic bradykinin substrate analog was approximately 15% of the wild type, while the H167A mutant had no detectable activity. Both Y226F and H167A were more susceptible to extensive degradation by trypsin compared with the wild-type protein. The zinc content in the wild-type and Y226F mutant proteins were similar, one molecule of zinc per subunit. The results indicate that Y226 is not essential for zinc binding, but Y226 and H167 are essential for full enzymatic activity and stability of the metalloproteinase.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey 17033-0850, USA.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Doll
authors.forename: James P
authors.initials: JP
authors.lastname: Villa
authors.forename: Faoud T
authors.initials: FT
authors.lastname: Ishmael
authors.forename: Judith S
authors.initials: JS
authors.lastname: Bond
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004795:Enzyme Stability; D007700:Kinetics; D008024:Ligands; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D015032:Zinc / Q000032:analysis / Q000737:chemistry
chemical_list: D008024:Ligands; D008666:Metalloendopeptidases; C053833:meprin A; C071288:astacin; D015032:Zinc