THIMET OLIGOPEPTIDASE - SITE-DIRECTED MUTAGENESIS DISPROVES PREVIOUS ASSUMPTIONS ABOUT THE NATURE OF THE CATALYTIC SITE

Zinc metallopeptidases that contain the His-Glu-Xaa-Xaa-His (HEXXH) mo tif generally have a third ligand of the metal ion that may be either a Glu residue (in clan MA) or a His residue (in clan MB) (Rawlings and Barrett (1995) Methods Enzymol, 248, 183-228). Thimet oligopeptidase has not yet been assigned to either dan, and both Glu and His residues have been proposed as the third ligand. We mutated candidate ligand r esidues in the recombinant enzyme and identified Glu, His and Asp resi dues that are important for catalytic activity and/or stability of the protein. However, neither of the Glu and His residues close to the HE XXH motif that have previously been suggested to be ligands is require d for the binding of zinc. We conclude that thimet oligopeptidase is n ot a member of dan MA or dan MB and it is likely that the enzyme posse sses a catalytic site and protein fold different from those identified in any metallopeptidase to date. The definitive identification of the third zinc ligand may well require the determination of the crystallo graphic structure of thimet oligopeptidase or one of its homologues. ( C) 1998 Federation of European Biochemical Societies.