CRYSTAL STRUCTURE OF THE ASPARTIC PROTEINASE FROM Rhizomucor miehei at 2.15 Å. Jian Yang^*, Alexei Teplyakov^# and J. Wilson Quail^*. ^*Department of Chemistry, University of Sasakatchewan, Saskatoon, Saskatchewan, Canada S7N 5C9, and ^#EMBL, Hamburg Outstation, Hamburg, Germany

The crystal structure of the aspartic proteinase from Rhizomucor miehei (RMP, EC. 3.4.23.23) has been refined to 2.15 Å resolution to a crystallographic R-factor of 21.5% and a R-free factor of 28.1%. The protein contains two domains, which consist predominantly of [[beta]]-sheets. The C-terminal domain is less rigid than the N-terminal domain due to the crystal packing. A large substrate binding cleft is clearly visible between the two domains and the catalytic residues Asp38 and Asp237 are located in the middle of the cleft with a water molecule bridging these two carboxyl groups. We will report the results of the surface electrostatic potential calculations of RMP and other aspartic proteinases' active sites that show the pH optimum of each aspartic proteinase is determined by the electrostatic potentials of the two carboxyl groups of the two catalytic aspartates. The electrostatic potentials of the two aspartates' carboxyl groups are, in turn, determined by the active-site environment, especially residues 19 and 332. The protein is glycosylated at Asp79 and Asp188. The glycosylations are believed to stabilize the protein by sterically inhibiting the attack of other proteinases and contributing to the protein's high thermal stability. Three-dimensional structure alignment and sequence alignment of RMP with other aspartic proteinases have shown that RMP is structurally similar to Mucor pusillus aspartic proteinase (MPP). RMP and MPP are as distinct from other fungal enzymes as they are from the mammalian enzymes. This suggests that RMP and MPP diverged from the main stream of aspartic proteinases at an early stage of evolution.

This research is funded by NSERC, Canada.