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

Yapsin 1, a novel aspartic protease with unique specificity for basic residues, was shown to cleave CCK13-33 at Lys(23). 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 part icular, the S2' subsite has three negative charges in and close to thi s pocket that can provide strong electrostatic interactions with a bas ic residue. The model, therefore, predicts that substrates with a basi c residue in the P1 position would be favored with additional basic re sidues binding to the other electronegative pockets. A deletion of six residues close to the S1 pocket in yapsin 1, relative to rhizopuspeps in and other aspartic proteases of known 3D structure, is likely to af fect its specificity. The model was tested using CCK13-33 analogues. W e report that yapsin 1 preferentially cleaves a CCK13-33 substrate wit h a basic residue in the pi position since the substrates with Ala in pi were not cleaved. Furthermore, the cleavage efficiency of yapsin 1 was enhanced for CCK13-33 analogues with arginine residues flanking th e P1 position. An alanine residue, substituting for the arginine resid ue 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 clea vage efficiency by 21-, 3- and 7-fold, respectively. Substitution of L ys(23) in CCK13-33 With arginine resulted not only in cleavage after t he substituted arginine residue, but also forced a cleavage after Met( 25), suggesting that an arginine residue in the S2' pocket is so favor able that it can affect the primary specificity of yapsin 1. These res ults are consistent with the predictions from the molecular model of y apsin 1.