Purification and characterization of human metallocarboxypeptidase Z

Carboxylseptidase Z (CPZ) is a recently discovered member of the metallocar boxypeptidase gene family that has an N-terminal domain related to the Wnt/ wingless binding domain or frizzled receptors and other proteins. To furthe r characterize the enzymatic properties of CPZ, the enzyme was purified usi ng Arg- and heparin-affinity columns. CPZ has a neutral pH optimum, and is inhibited by chelating agents and several divalent cations (Zn2+, Mn2+, Cd2 +, Cu2+, Hg2+). Active site-directed inhibitors of several other metallocar boxypeptidases also-inhibit CPZ activity with moderate potency. CPZ cleaves substrates with C-terminal Arg;residues, preferring peptides with an Ala i n the penultimate position. No activity is detected toward substrates with an Ile-Arg or a Pro-Arg sequence. The Km for dansyl-Phe-Ala-Arg and dansyl- Pro-Ala-Arg are both approximately 2 mM. Taken together, these data suggest s a;selective role for CPZ in the processing of extracellular peptides or p roteins. (C) 1999 Academic Press.