Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of polyamine synthesis are recycled in a novel pathway in which 5 -methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5 -methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5 -methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not been described previously. 5 -Methylthio-immucillin-H, a transition state analogue inhibitor that is selective for malarial relative to human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may also have application as anti-malarials.

Recombinant human thymidine phosphorylase catalyzes the reaction of arsenate with thymidine to form thymine and 2-deoxyribose 1-arsenate, which rapidly decomposes to 2-deoxyribose and inorganic arsenate. The transition-state structure of this reaction was determined using kinetic isotope effect analysis followed by computer modeling. Experimental kinetic isotope effects were determined at physiological pH and 37 degrees C. The extent of forward commitment to catalysis was determined by pulse-chase experiments to be 0.70%. The intrinsic kinetic isotope effects for [1'-(3)H]-, [2'R-(3)H]-, [2'S-(3)H]-, [4'-(3)H]-, [5'-(3)H]-, [1'-(14)C]-, and [1-(15)N]-thymidines were determined to be 0.989 +/- 0.002, 0.974 +/- 0.002, 1.036 +/- 0.002, 1.020 +/- 0.003, 1.061 +/- 0.003, 1.139 +/- 0.005, and 1.022 +/- 0.005, respectively. A computer-generated model, based on density functional electronic structure calculations, was fit to the experimental isotope effect. The structure of the transition state confirms that human thymidine phosphorylase proceeds through an S(N)2-like transition state with bond orders of 0.50 to the thymine leaving group and 0.33 to the attacking oxygen nucleophile. The reaction differs from the dissociative transition states previously reported for N-ribosyl transferases and is the first demonstration of a nucleophilic transition state for an N-ribosyl transferase. The large primary (14)C isotope effect of 1.139 can occur only in nucleophilic displacements and is the largest (14)C primary isotope effect reported for an enzymatic reaction. A transition state structure with substantial bond order to the attacking nucleophile and leaving group is confirmed by the slightly inverse 1'-(3)H isotope effect, demonstrating that the transition state is compressed by the impinging steric bulk of the nucleophile and leaving group.

The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with bound inosine or transition state analogues show His 257 within hydrogen-bonding distance to the 5′-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for Immucillin-H (ImmH), binding this mimic of an early transition state as much as 370-fold (K m /K i ) less tightly than native PNP. In contrast, these mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as well as the native enzyme. These results indicate that His 257 serves an important role in the early stages of transition state formation. Whereas mutation of His 257 resulted in little variation in the PNP·DADMe-ImmH·SO 4 structures, His257Phe·ImmH·PO 4 showed distortion at the 5′-hydroxyl, indicating the importance of H-bonding in positioning this group during progression to the transition state. Binding isotope effect (BIE) and kinetic isotope effect (KIE) studies on the remote 5′-3 H for the arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a moderate electronic distortion toward the transition state in the Michaelis complex with continued development of a similar distortion at the transition state. The mutants His257Phe, His257Gly, and His257Asp altered the 5′-3 H intrinsic KIE to −3%, −14%, and 7%, respectively, while the BIEs contributed 2%, 2%, and −2%, respectively. These surprising results establish that forces in the Michaelis complex, reported by the BIEs, can be reversed or enhanced at the transition state. Keywordspurine nucleoside phosphorylase; mutagenesis; binding isotope effect; kinetic isotope effect; transition state; binding distortion; transition state geometry; dynamics Determination of the transition state structure of enzymatic reactions allows for the design of tight binding transition state analogue inhibitors. These transition state mimics can subvert the energetics that govern formation of the enzymatic transition state to achieve binding orders of magnitude stronger than that of substrate. The transition state structures of enzymatic reactions have frequently been established by measuring kinetic isotope effects (KIEs 1 ) from the competitive reaction of isotopically labeled substrates. These isotope effects on k cat /K m , often † Supported by NIH Research Grant GM41916.* To whom correspondence should be addressed. vern@aecom.yu.edu; Telephone, (718) 430-2813; Fax, (718) . ‡ Current address: Department of Chemistry, Barnard College, 3009 Broadway, New York, NY 10027. § Current address: Center for Synchrotron Bioscience, Brookhaven National Laboratory, Upton, NY 11973.1 Abbreviations: KIE, kinetic isotope effect; V/K KIE, kinetic isotope effect on the second-order rate constant, k cat /K m ; BIE, binding isotope effect; HsPNP, human purine nucleoside phosphorylase; PDB, Protein Data Bank; ImmH, Immucillin-H; DADMe-ImmH, 4′-deaza-1′-aza-2′-deoxy-1′-(9-methylene)-Immucillin-H; RMS, root mea...

The relationship between 3-deoxy-D-manno-2-octulosonic acid 8-phosphate (KDO 8-P) synthase and 3-deoxy-D-arabino-2-heptulosonic acid 7-phosphate (DAH 7-P) synthase has not been adequately addressed in the literature. Based on recent reports of a metal requiring KDO 8-P synthase and the newly solved X-ray crystal structures of both Escherichia coli KDO 8-P synthase and DAH 7-P synthase, we begin to address the evolutionary kinship between these catalytically similar enzymes. Using a maximum likelihood-based grouping of 29 KDO 8-P synthase sequences, we demonstrate the existence of a new class of KDO 8-P synthase, the members of which we propose to require a metal cofactor for catalysis. Similarly, we hypothesize a class of DAH 7-P synthase that does not have the metal requirement of the heretofore model E. coli enzyme. Based on this information and a careful investigation of the reported X-ray crystal structures, we also propose that KDO 8-P synthase and DAH 7-P synthase are the product of a divergent evolutionary process from a common ancestor.