Transition state analogue inhibitors of purine nucleoside phosphorylase from Plasmodium falciparum.
Immucillins are logically designed transition-state analogue inhibitors of mammalian purine nucleoside phosphorylase (PNP) that induce purine-less death of Plasmodium falciparum in cultured erythrocytes (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226-3231). PNP is present at high levels in human erythrocytes and in P. falciparum, but the Plasmodium enzyme has not been characterized. A search of the P. falciparum genome data base yielded an open reading frame similar to the PNP from Escherichia coli. PNP from P. falciparum (P. falciparum PNP) was cloned, overexpressed in E. coli, purified, and characterized. The primary amino acid sequence has 26% identity with E. coli PNP, has 20% identity with human PNP, and is phylogenetically unique among known PNPs with equal genetic distance between PNPs and uridine phosphorylases. Recombinant P. falciparum PNP is catalytically active for inosine and guanosine but is less active for uridine. The immucillins are powerful inhibitors of P. falciparum PNP. Immucillin-H is a slow onset tight binding inhibitor with a K(i)* value of 0.6 nm. Eight related immucillins are also powerful inhibitors with dissociation constants from 0.9 to 20 nm. The K(m)/K(i)* value for immucillin-H is 9000, making this inhibitor the most powerful yet reported for P. falciparum PNP. The PNP from P. falciparum differs from the human enzyme by a lower K(m) for inosine, decreased preference for deoxyguanosine, and reduced affinity for the immucillins, with the exception of 5'-deoxy-immucillin-H. These properties of P. falciparum PNP are consistent with a metabolic role in purine salvage and provide an explanation for the antibiotic effect of the immucillins on P. falciparum cultured in human erythrocytes.
Kicska
Gregory A
GA
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Tyler
Peter C
PC
Evans
Gary B
GB
Furneaux
Richard H
RH
Kim
Kami
K
Schramm
Vern L
VL
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004912:Erythrocytes / Q000469:parasitology; D004926:Escherichia coli / Q000201:enzymology; D018503:Genome, Protozoan; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D011683:Purine-Nucleoside Phosphorylase