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Active site specificity of plasmepsin II
J Westling1, P Cipullo, S H Hung
1Department of Biochemistry & Molecular Biology, College of Medicine, University of Florida, Gainesville 32610-0245, USA.
Protein Science : a Publication of the Protein Society
|November 5, 1999
Summary
Researchers studied plasmepsin II, an enzyme from the malaria parasite Plasmodium falciparum, to understand its substrate specificity. Mutagenesis revealed key amino acids enabling cleavage of specific peptide substrates, aiding in drug development.
Area of Science:
- Biochemistry
- Enzymology
- Parasitology
Background:
- Aspartic proteinases share similar structures and mechanisms but exhibit unique substrate specificities.
- These specificities are determined by amino acid variations within the enzyme's active site.
- Plasmepsin II, from Plasmodium falciparum, is a key target for antimalarial drug development due to its role in parasite survival.
Purpose of the Study:
- To elucidate the substrate binding preferences of plasmepsin II.
- To identify specific amino acid residues responsible for plasmepsin II's unique specificity.
- To engineer improved substrates and potentially inhibitors for plasmepsin II.
Main Methods:
- Systematic analysis of chromogenic octapeptides with substitutions to determine binding preferences.
- X-ray crystallography to analyze the three-dimensional structure of plasmepsin II.
- Site-directed mutagenesis of specific amino acid residues (Met13, Ser77, Ile287) within the active site.
Main Results:
- Designed novel, improved substrates for plasmepsin II (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys).
- Crystal structure analysis provided insights into enzyme-substrate interactions.
- Mutations Met13Glu and Ile287Glu (single and double) conferred the ability to cleave substrates containing Lys residues.
Conclusions:
- Specific amino acid residues, particularly Met13 and Ile287, are critical for plasmepsin II's substrate specificity.
- Understanding these interactions allows for the rational design of targeted antimalarial therapies.
- The engineered mutants demonstrate the plasticity of the active site and potential for altering enzyme function.