THE 3-DIMENSIONAL STRUCTURE AT 2.4 ANGSTROM RESOLUTION OF GLYCOSYLATED PROTEINASE-A FROM THE LYSOSOME-LIKE VACUOLE OF SACCHAROMYCES-CEREVISIAE

The crystal structures of glycosylated native proteinase A, an asparti c proteinase found in the vacuole of Saccharomyces cerevisiae, and its complex with a difluorostatone-containing tripeptide have been determ ined by molecular replacement to 3.5 Angstrom and 2.4 Angstrom resolut ions, respectively. Superposition of the bound and native forms gave a n r.m.s. difference of 0.6 Angstrom largely reflecting the poor resolu tion of the native crystal structure. The secondary and tertiary struc tures are highly similar to those found in porcine pepsin and lysosoma l cathepsin D; superposition of the structure of proteinase A bound to the difluorostatone inhibitor on those of pepsin and cathepsin D gave pairwise r.m.s. differences for C-alpha atoms of 1.36 Angstrom and 0. 88 Angstrom. Most differences occur in loop regions. Comparison of the structure of the proteinase A-difluorostatone complex with that of en dothiapepsin bound with the same inhibitor shows that the conformation and hydrogen bond interactions of the inhibitor in the active site ar e very similar, even though the enzymes have only 27% sequence identit y. Electron density for the crystal structure of the proteinase A comp lex reveals five residues of the oligosaccharide structure attached to Asn67: Man-(1 --> 2)-alpha-Man-(1 --> 3)-beta-Man-(1 --> 4)-beta-GlcN Ac-(1 --> 4)-beta-GlcNAc-Asn-67. The first three residues of the oligo saccharide cover the same region of the protein surface as those of th e oligosaccharide attached to the equivalent position in cathepsin D. The second carbohydrate attachment site is disordered beyond the first carbohydrate residue in both enzymes. (C) 1997 Academic Press Limited .