Abstract
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.
Highlights
EXPERIMENTAL PROCEDURESReagents—Imm-H ((1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy1,4-imino-D-ribitol) and other immucillins were synthesized from Dgulonolactone and chemically protected 9-deazahypoxanthine [33]
Plasmodium falciparum is responsible for the majority of deaths due to malaria [1]
The reading frame for P. falciparum purine nucleoside phosphorylase (PNP) was amplified using Polymerase chain reaction (PCR) and DNA sequencing of the product showed no difference from the data base sequence, except for the changes introduced at the N terminus
Summary
Reagents—Imm-H ((1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy1,4-imino-D-ribitol) and other immucillins were synthesized from Dgulonolactone and chemically protected 9-deazahypoxanthine [33]. Catalytic Activity and Determination of Kinetic Constants—Partially purified P. falciparum PNP was used for kinetic and inhibition studies (60 –70% as determined by denaturing polyacrylamide gel electrophoresis), some of which were repeated with highly purified enzyme. The results with both preparations were the same. In a coupled assay containing alkaline phosphatase, the reaction was followed by measuring formation of adenine from adenosine or 5Ј-methylthioadenosine (E256 ϭ 1.9 mMϪ1 cmϪ1). Coupled assay containing alkaline phosphatase, the reaction was followed by monitoring the formation of guanine from guanosine or deoxyguanosine (E258 ϭ 5.2 mMϪ1 cmϪ1 [29]). The observed rate vs is the rate following slow-onset inhibition, and kcat is the uninhibited rate at saturating substrate concentration
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
