Plasmepsin II is one of the four catalytically active plasmepsins found in the food vacuole of Plasmodium falciparum. These enzymes initiate hemoglobin degradation by cleavage at the -chain between Phe33 and Leu34. The crystal structures of Ser205 mutant plasmepsin II from P. falciparum in complex with two inhibitors have been re®ned at a resolution of 1.8 A Ê in the space group I222 and to R factors of 19.9 and 19.5%. Each crystal contains one monomer in the asymmetric unit. Both inhibitors have a Phe± Leu core and incorporate tetrahedral transition-state mimetic hydroxypropylamine. The inhibitor rs367 possesses a 2,6-dimethylphenyloxyacetyl group at the P2 position and 3-aminobenzamide at the P2 H position, while rs370 has the same P2 group but 4-aminobenzamide in the P2 H position. These complexes reveal key conserved hydrogen bonds between the inhibitor and the binding-cavity residues, notably with the¯ap residues Val78 and Ser79, the catalytic dyad Asp34 and Asp214 and the residues Ser218 and Gly36 that are in proximity to the catalytic dyad. The structures also show unexpected conformational variability of the binding cavity of plasmepsin II and may re¯ect the mode of binding of the hemoglobin -chain for cleavage.

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Two new potent allosteric effectors of hemoglobin, RSR-4 [2-[4-[[(3,5-dichloroanilino)carbonyl]-methyl]phenoxy]-2- methylpropionic acid] and RSR-13 [2-[4-[[(3,5-dimethlanilino)carbonyl]methyl]-phenoxy]-2-methylp rop ionic, are compared to the previously reported compounds L3,5 and L3,4,5 [Lalezari, I., Lalezari, P., Poyart, C., Marden, M., Kister, J., Bohn, B., Fermi, G., & Perutz, M. F. (1990) Biochemistry 29, 1515]. Unlike L3,5 and L3,4,5, RSR-4 and RSR-13 are less impeded by physiological concentrations of serum albumin. RSR-4 has also been shown to be more effective than L3,5 in shifting the allosteric equilibrium of bovine Hb toward the low-affinity T-state. X-ray crystal studies show that both RSR-4 and RSR-13 bind to only one pair of symmetry-related sites in the Hb central water cavity whereas previous studies on L3,5 and L3,4,5 demonstrated a second pair of symmetry-related binding sites near Arg 104 beta. Three major interactions between these allosteric effectors and Hb include the acid group with the guanidinium group of C-terminal Arg 141 alpha, the effector's amide oxygen with the ammonium ion of Lys 99 alpha, and the phi electrons of the halogenated or methylated aromatic ring and Asn 108 beta. No explanation has been found for the difference in number of binding sites observed for RSR-4 and RSR-13 (two sites) compared to L3,5 and L3,4,5 (four sites); also no correlation has been made between the number of binding sites and degree of allosteric shift in the oxygen equilibrium curve.(ABSTRACT TRUNCATED AT 250 WORDS)