Crystallographic studies of Rhizomucor miehei aspartic proteinase and its pepstatin A complex

The crystal structures of Rhizomucor miehei aspartic proteinase (RMP) and its pepstatin A complex have been determined at 2.15 Å and 2.7 Å, respectively. The structure of the native enzyme was refined to a crystallographic R-factor of 21.5% (R-free = 28.1%). RMP contains two domains that consist predominantly of β-sheets. A large substrate binding cleft is clearly visible between the two domains, and the two catalytic residues Asp38 and Asp237 are located in the middle of the cleft with a water molecule bridging the carboxyl groups of Asp38 and Asp237. It is proposed that the optimal pH of each aspartic proteinase is determined by the electrostatic potential at the active site, which, in turn, is determined by the positions and orientations of all the residues near the active site. RMP is the most glycosylated and most thermally stable among the aspartic proteinases. It is proposed that the highly flexible carbohydrates act as heat reservoirs to stabilize the conformation of RMP and thereby provide the enzyme with high thermal stability. Three-dimensional structural and sequence alignments of RMP with other aspartic proteinases suggest that RMP and Mucor pusillus aspartic proteinase (MPP) diverged from the main stream of aspartic proteinases at an early stage of evolution. The structure of the RMP-pepstatin A complex was refined to a crystallographic R-value of 19.3% (R-free = 28.0%). In the final model, a pepstatin A molecule fits into the large substrate-binding cleft between the two domains of RMP in an extended conformation up to the alanine residue at the P2' position. The statine residue at the "P3'-P4'" position forms an inverse ɣ-turn (P3' - P1'), with its leucyl side chain binding into the S1' subsite. The inhibitor interacts with the residues of the substrate binding pocket by either hydrogen bonds or hydrophobic interactions, or both