Synthesis and characterization of the first potent inhibitor of yapsin 1. Implications for the study of yapsin-like enzymes.
The potent peptidic inhibitor, Y1, of the basic residue-specific yeast aspartyl protease, yapsin 1, was synthesized and characterized. The inhibitor was based on the peptide sequence of a cholecystokinin(13-33) analog that yapsin 1 cleaved with an efficiency of 5.2 x 10(5) m(-1) s(-1) (Olsen, V., Guruprasad, K., Cawley, N. X., Chen, H. C., Blundell, T. L., and Loh, Y. P. (1998) Biochemistry 37, 2768-2777). The apparent K(i) of Y1 for the inhibition of yapsin 1 was determined to be 64.5 nm, and the mechanism is competitive. Y2 was also developed as an analog of Y1 for coupling to agarose beads. The resulting inhibitor-coupled agarose beads were successfully used to purify yapsin 1 to apparent homogeneity from conditioned medium of a yeast expression system. Utilization of this new reagent greatly facilitates the purification of yapsin 1 and should also enable the identification of new yapsin-like enzymes from mammalian and nonmammalian sources. In this regard, Y1 also efficiently inhibited Sap9p, a secreted aspartyl protease from the human pathogen, Candida albicans, which has specificity for basic residues similar to yapsin 1 and might provide the basis for the prevention or control of its virulence. A single-step purification of Sap9p from conditioned medium was also accomplished with the inhibitor column. N-terminal amino acid sequence analysis yielded two sequences indicating that Sap9p is composed of two subunits, designated here as alpha and beta, similar to yapsin 1.
Cawley
Niamh X
NX
Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. cawleyn@mail.nih.gov
Chino
Masao
M
Maldonado
Alex
A
Rodriguez
Yazmin M
YM
Loh
Y Peng
YP
Ellman
Jonathan A
JA
D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004745:Enkephalins / Q000037:antagonists & inhibitors; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D011480:Protease Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology*; D011498:Protein Precursors / Q000037:antagonists & inhibitors; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D012044:Regression Analysis; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship
D004745:Enkephalins; D011480:Protease Inhibitors; D011498:Protein Precursors; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; C029992:proenkephalin; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases; C499266:YPS1 protein, S cerevisiae