title: Energetic mapping of transition state analogue interactions with human and Plasmodium falciparum purine nucleoside phosphorylases.
abstract: Human purine nucleoside phosphorylase (huPNP) is essential for human T-cell division by removing deoxyguanosine and preventing dGTP imbalance. Plasmodium falciparum expresses a distinct PNP (PfPNP) with a unique substrate specificity that includes 5'-methylthioinosine. The PfPNP functions both in purine salvage and in recycling purine groups from the polyamine synthetic pathway. Immucillin-H is an inhibitor of both huPNP and PfPNPs. It kills activated human T-cells and induces purine-less death in P. falciparum. Immucillin-H is a transition state analogue designed to mimic the early transition state of bovine PNP. The DADMe-Immucillins are second generation transition state analogues designed to match the fully dissociated transition states of huPNP and PfPNP. Immucillins, DADMe-Immucillins and related analogues are compared for their energetic interactions with human and P. falciparum PNPs. Immucillin-H and DADMe-Immucillin-H are 860 and 500 pM inhibitors against P. falciparum PNP but bind human PNP 15-35 times more tightly. This common pattern is a result of kcat for huPNP being 18-fold greater than kcat for PfPNP. This energetic binding difference between huPNP and PfPNP supports the k(chem)/kcat binding argument for transition state analogues. Preferential PfPNP inhibition is gained in the Immucillins by 5'-methylthio substitution which exploits the unique substrate specificity of PfPNP. Human PNP achieves part of its catalytic potential from 5'-OH neighboring group participation. When PfPNP acts on 5'-methylthioinosine, this interaction is not possible. Compensation for the 5'-OH effect in the P. falciparum enzyme is provided by improved leaving group interactions with Asp206 as a general acid compared with Asn at this position in huPNP. Specific atomic modifications in the transition state analogues cause disproportionate binding differences between huPNP and PfPNPs and pinpoint energetic binding differences despite similar transition states.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Lewandowicz
authors.forename: Erika A Taylor
authors.initials: EA
authors.lastname: Ringia
authors.forename: Li-Min
authors.initials: LM
authors.lastname: Ting
authors.forename: Kami
authors.initials: K
authors.lastname: Kim
authors.forename: Peter C
authors.initials: PC
authors.lastname: Tyler
authors.forename: Gary B
authors.initials: GB
authors.lastname: Evans
authors.forename: Olga V
authors.initials: OV
authors.lastname: Zubkova
authors.forename: Simon
authors.initials: S
authors.lastname: Mee
authors.forename: Gavin F
authors.initials: GF
authors.lastname: Painter
authors.forename: Dirk H
authors.initials: DH
authors.lastname: Lenz
authors.forename: Richard H
authors.initials: RH
authors.lastname: Furneaux
authors.forename: Vern L
authors.initials: VL
authors.lastname: Schramm
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D002417:Cattle; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010963:Plasmodium falciparum / Q000201:enzymology*; D011073:Polyamines / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011684:Purine Nucleosides; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000378:metabolism; D011687:Purines / Q000737:chemistry; D011744:Pyrimidinones / Q000737:chemistry; D011758:Pyrroles / Q000737:chemistry; D055672:Static Electricity; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000378:metabolism
chemical_list: D011073:Polyamines; D011684:Purine Nucleosides; D011687:Purines; D011744:Pyrimidinones; D011758:Pyrroles; D001224:Aspartic Acid; C113101:forodesine; D011683:Purine-Nucleoside Phosphorylase; C030985:purine