Exam3_paper1 - Biochemistry 2007, 46, 12405-12415 12405...

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Anopheles gambiae Purine Nucleoside Phosphorylase: Catalysis, Structure, and Inhibition ²,‡ Erika A. Taylor, § Agnes Rinaldo-Matthis, Lei Li, Mahmoud Ghanem, Keith Z. Hazleton, Marı´a B. Cassera, Steven C. Almo, and Vern L. Schramm* Department of Biochemistry, Albert Einstein College of Medicine at Yeshi V a Uni V ersity, 1300 Morris Park A V enue, Bronx, New York 10461 Recei V ed May 25, 2007; Re V ised Manuscript Recei V ed August 15, 2007 ABSTRACT : The purine salvage pathway of Anopheles gambiae , a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has k cat values of 54 and 41 s - 1 for 2 -deoxyinosine and inosine, its preferred substrates, and 1.0 s - 1 for guanosine. However, the chemical step is fast for AgPNP at 226 s - 1 for guanosine in pre-steady-state studies. 5 -Deaza-1 -aza-2 -deoxy-1 -(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2 -deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable K m / K i *of5 .4 × 10 7 , and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 Å to reveal the differences in substrate and inhibitor specificity. The distance from the N1 cation to the phosphate O4 anion is shorter in the AgPNP DADMe-ImmH PO 4 complex than in HsPNP DADMe-ImmH SO 4 , offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP. Malaria is one of the oldest known human diseases, first described in the fifth century B.C. by Hippocrates ( 1 ). It was then, as it is now, a seasonal disease of the poor, being one of the principal causes of mortality in Africa, southeast Asia, and Latin America ( 2 ). Current World Health Organization figures estimate that malaria infection causes 300 million cases of acute illnesses and at least one million deaths annually, primarily of children under 5 years of age ( 2 ). In addition to the human toll, the economic costs have been estimated as 1% of world gross domestic product (GDP) per year ( 3 ). Attempts to break the cycle of malaria transmission include mosquito control, bed nets, antibiotics, and education, but the lack of consistent utilization of these methods has
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This note was uploaded on 07/23/2008 for the course CHEM 476 taught by Professor Bevilacqua,philip during the Fall '07 term at Pennsylvania State University, University Park.

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Exam3_paper1 - Biochemistry 2007, 46, 12405-12415 12405...

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