Active site specificity of plasmepsin II

Members of the aspartic proteinase family of enzymes have very similar thre e-dimensional structures and catalytic mechanisms. Each, however, has uniqu e substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of t he aspartic proteinase class from the malaria parasite, Plasmodium falcipar um, chromogenic octapeptides having systematic substitutions at various pos itions in the sequence were analyzed. This enabled the design of new, impro ved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, wh ere * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specifi c amino acids (Met13, Ser77, and Ile287) that were suspected of contributin g to active site binding and specificity were chosen for site-directed muta genesis experiments. The Met13Glu and Ile287Glu single mutants and the Met1 3Glu/Ile287Glu double mutant gain the ability to cleave substrates containi ng Lys residues.