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Yeast Prohormone Processing Enzyme (KEX2 Gene Product) is a Ca2+-Dependent Serine Protease
Robert S. Fuller, Anthony Brake and Jeremy Thorner
Proceedings of the National Academy of Sciences of the United States of America
Vol. 86, No. 5 (Mar. 1, 1989), pp. 1434-1438
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/33209
Page Count: 5
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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 detergents; (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) unaffected by 5-10 mM phenylmethylsulfonyl fluoride, Nα-(p-tosyl)lysine chloromethyl ketone, or L-1-tosylamido-2-phenylethyl chloromethyl ketone, but inactivated by 1-2 μ M Ala-Lys-Arg-chloromethyl ketone; (v) labeled specifically by 125I-labeled Tyr-Ala-Lys-Arg-chloromethyl ketone; and (vi) resistant 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 125I-labeled Tyr-Ala-Lys-Arg-chloromethyl ketone.
Proceedings of the National Academy of Sciences of the United States of America © 1989 National Academy of Sciences