Zinc Ligands in an Astacin Family Metalloprotease Meprin A

• Published in: Biological chemistry Vol. 383; no. 7-8; pp. 1167 - 1173
• Main Authors: Doll, B. A., Villa, J. P., Ishmael, F. T., Bond, J. S.
• Format: Journal Article
• Language: English
• Published: Germany Walter de Gruyter 01.07.2002
• Subjects: Animals; Binding Sites - genetics; Conserved Sequence; Enzyme Stability; Kinetics; Ligands; Metalloendopeptidases - chemistry; Metalloendopeptidases - genetics; Metalloendopeptidases - metabolism; Mice; Mutagenesis, Site-Directed; Mutation, Missense; Zinc - analysis; Zinc - chemistry
• ISSN: 1431-6730; 1437-4315
• DOI: 10.1515/BC.2002.128

A conserved tyrosine residue in the astacin family of metalloproteases is one of five ligands proposed to coordinate zinc at the active site. Sitedirected mutagenesis of the conserved Tyr (Y226) of recombinant mouse meprin α 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 α 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 noncovalentlylinked 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 wildtype protein. The zinc content in the wildtype 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.