A/B-type metallocarboxypeptidases (MCPs) are among the most thoroughly studied proteolytic enzymes, and their catalytic mechanisms have been considered as prototypes even for several unrelated metalloprote(in)ase families. It has long been postulated that the nature of the side chains of at least five substrate residues, i.e., P4-P1′, influence K m and kcat and that once the peptide or protein substrate is cleaved, both products remain in the first instance bound to the active-site cleft of the enzyme in a double-product complex. Structural details of binding of substrate to the nonprimed side of the cleft have largely relied on complexes with protein inhibitors and peptidomimetic small-molecule inhibitors that do not span the entire groove. In the former, the presence of N-terminal globular protein domains participating in large-scale interactions with the surface of the cognate catalytic domain outside the active-site cleft mostly conditions the way their C-terminal tails bind to the cleft. Accordingly, they may not be accurate models for a product complex. We hereby provide the structural details of a true cleaved double-product complex with a hexapeptide of an MCP engaged in prostate cancer, human carboxypeptidase A4, employing diffraction data to 1.6 Å resolution (Rcryst and Rfree = 0.159 and 0.176, respectively). These studies provide detailed information about subsites S 5-S1′ and contribute to our knowledge of the cleavage mechanism, which is revisited in light of these new structural insights. © 2007 American Chemical Society.
|Publication status||Published - 12 Jun 2007|