Cleavage Efficiency of the Novel Aspartic Protease Yapsin 1 (Yap3p) Enhanced for Substrates with Arginine Residues Flanking the P1 Site:  Correlation with Electronegative Active-Site Pockets Predicted by Molecular Modeling^,

Abstract

Yapsin 1, a novel aspartic protease with unique specificity for basic residues, was shown to cleave CCK13-33 at Lys23. Molecular modeling of yapsin 1 identified the active-site cleft to have negative residues close to or within the S6, S3, S2, S1, S1', S2', and S3' pockets and is more electronegative than rhizopuspepsin or endothiapepsin. In particular, the S2' subsite has three negative charges in and close to this pocket that can provide strong electrostatic interactions with a basic residue. The model, therefore, predicts that substrates with a basic residue in the P1 position would be favored with additional basic residues binding to the other electronegative pockets. A deletion of six residues close to the S1 pocket in yapsin 1, relative to rhizopuspepsin and other aspartic proteases of known 3D structure, is likely to affect its specificity. The model was tested using CCK13-33 analogues. We report that yapsin 1 preferentially cleaves a CCK13-33 substrate with a basic residue in the P1 position since the substrates with Ala in P1 were not cleaved. Furthermore, the cleavage efficiency of yapsin 1 was enhanced for CCK13-33 analogues with arginine residues flanking the P1 position. An alanine residue, substituting for the arginine residue in the P6 position in CCK13-33, resulted in a 50% reduction in the cleavage efficiency. Substitution with arginine residues downstream of the cleavage site at the P2', P3', or P6' position increased the cleavage efficiency by 21-, 3- and 7-fold, respectively. Substitution of Lys23 in CCK13-33 with arginine resulted not only in cleavage after the substituted arginine residue, but also forced a cleavage after Met25, suggesting that an arginine residue in the S2' pocket is so favorable that it can affect the primary specificity of yapsin 1. These results are consistent with the predictions from the molecular model of yapsin 1.

Proteolytic Cleavage of Covalently Linked Cell Wall Proteins by Candida albicans Sap9 and Sap10

“…The near neutral pH optima and activity over a broad pH range distinguishes Sap9 and Sap10 from the other secreted Saps, which exhibit activity at more acidic pH optima and a more narrow pH range. The S. cerevisiae yapsins show similar activity over a broad pH range, depending on the substrate cleaved (20,52), but the significant activity at neutral to basic pH seems to be specific for Sap9 and Sap10. This may reflect the host-associated lifestyle of C. albicans requiring regulatory proteolytic digests at several different host niches with different pH values.…”

Endocrinomic profile of neurointermediate lobe pituitary prohormone processing in PC1/3- and PC2-Null mice using SELDI-TOF mass spectrometry

“…Some prior studies on prohormone processing have concentrated on the use of synthetic peptides and purified processing enzymes for in vitro analysis (Cawley et al 1996, Coates & Birch 1998, Olsen et al 1998. Other studies have used over-expression of substrate and enzyme in a cell system (Rouille et al 1992, Cool et al 1996, Wang et al 1998, Min et al 1999.…”

Glycosylphosphatidylinositol-anchored Proteases of Candida albicans Target Proteins Necessary for Both Cellular Processes and Host-Pathogen Interactions

“…Digests were similar to the activity of S. cerevisiae yapsins or Kex2-regulatory proteases with hydrolysis at KR, KK, or single Lys (K) sites (Table 1). Sap9 and Sap10 preferred cleavage after dibasic (KR, KK) or monobasic (Lys, Arg) residues, similar to yapsin 1 and 2, whereas the Kex2 protease cleaves only C-terminally to clusters of dibasic residues (13,29,30). Addition of uncharged Asn into the P1Ј side in the peptide PISKRNERS abolished cleavage by Sap9 but maintained the processing with Sap10 (Table 1).…”

“…P. pastoris transformation, and production of recombinant enzymes was described previously (29). Culture supernatants were harvested and dialyzed against 20 mM sodium citrate buffer, pH 6.5, containing 150 mM NaCl (buffer A).…”