Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease (prohormone convertase/membrane-bound protease/precursor maturation/secretory pathway/posttranslational modification)

The KEX2-encoded endoprotease was over- produced in yeast several hundred-fold and further purified to achieve a 10,000-fold enrichment in specific activity. The enzyme was (i) membrane-bound, but solubilized by deter- gents; (ii) able to cleave peptide substrates at both Lys-Arg and Arg-Arg sites; (iii) inhibited by EDTA and EGTA (but not o-phenanthroline), but fully reactivated by Ca2+; (iv) unaf- fected by 5-10 mM phenylmethylsulfonyl fluoride, Na-(p- tosyl)lysine chloromethyl ketone, or L-1-tosylamido-2-phenyl- ethyl chloromethyl ketone, but inactivated by 1-2 ,uM Ala- Lys-Arg-chloromethyl ketone; (v) labeled specifically by 125i. labeled Tyr-Ala-Lys-Arg-chloromethyl ketone; and (vi) resist- ant to trans-epoxysuccinate compounds (which inactivate thiol proteases), but inactivated by diisopropyl fluorophosphate (a diagnostic serine protease inhibitor). Mutant enzyme molecules lacking as many as 200 C-terminal residues still retained Ca2+-dependent protease activity and were labeled by 1211_ labeled Tyr-Ala-Lys-Arg-chloromethyl ketone.