Active-site specificity of digestive aspartic peptidases from the four species of Plasmodium that infect humans using chromogenic combinatorial peptide libraries.
Two targeted chromogenic octapeptide combinatorial libraries, comprised of 38 pools each containing 361 different peptides, were used to analyze the enzyme/substrate interactions of five plasmepsins. The first library (P1 library) was based on a good synthetic aspartic peptidase substrate [Westling, J., Cipullo, P., Hung, S. H., Saft, H., Dame, J. B., and Dunn, B. M. (1999) Protein Sci. 8, 2001-2009; Scarborough, P. E., and Dunn, B. M. (1994) Protein Eng. 7, 495-502] and had the sequence Lys-Pro-(Xaa)-Glu-P1*Nph-(Xaa)-Leu. The second library (P1' library) incorporated results with the plasmepsins from the first library and had the sequence Lys-Pro-Ile-(Xaa)-Nph*P1'-Gln-(Xaa). In both cases, P1 and P1' were fixed residues for a given peptide pool, where Nph was a para-nitrophenylalanine chromogenic reporter and Xaa was a mixture of 19 different amino acids. Kinetic assays monitoring the rates of cleavage of these libraries revealed the optimal P1 and P1' residues for the five plasmepsins as hydrophobic substitutions. Extended specificity preferences were obtained utilizing liquid chromatography-mass spectrometry (LC-MS) to analyze the cleavage products produced by enzyme-catalyzed digestion of the best pools of each peptide library. LC-MS analysis of the P1-Phe and P1'-Phe pools revealed the favored amino acids at the P3, P2, P2', and P3' positions. These analyses have provided new insights on the binding preferences of malarial digestive enzymes that were used to design specific methyleneamino peptidomimetic inhibitors of the plasmepsins. Some of these compounds were potent inhibitors of the five plasmepsins, and their possible binding modes were analyzed by computational methods.
Beyer
Bret B
BB
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida 32610, USA.
Johnson
Jodie V
JV
Chung
Alfred Y
AY
Li
Tang
T
Madabushi
Amrita
A
Agbandje-McKenna
Mavis
M
McKenna
Robert
R
Dame
John B
JB
Dunn
Ben M
BM
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000378:metabolism*; D001665:Binding Sites; D002863:Chromogenic Compounds / Q000737:chemistry / Q000378:metabolism*; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D019295:Computational Biology / Q000379:methods; D003198:Computer Simulation; D006868:Hydrolysis; D008288:Malaria / Q000201:enzymology*; D008958:Models, Molecular; D019151:Peptide Library; D010963:Plasmodium falciparum / Q000201:enzymology; D010965:Plasmodium malariae / Q000201:enzymology; D041122:Plasmodium ovale / Q000201:enzymology; D010966:Plasmodium vivax / Q000201:enzymology; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D015800:Protozoan Proteins; D013379:Substrate Specificity
D002863:Chromogenic Compounds; D019151:Peptide Library; D011480:Protease Inhibitors; D015800:Protozoan Proteins; D016282:Aspartic Acid Endopeptidases; C460085:signal peptide peptidase; C089643:plasmepsin; C100637:plasmepsin II