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Protein motions that occur on the microsecond to millisecond time scale have been linked to enzymatic rates observed for catalytic turnovers, but not to transition-state barrier crossing. It has been hypothesized that enzyme motions on the femtosecond time scale of bond vibrations play a role in transition state formation. Here, we perturb femtosecond motion by substituting all nonexchangeable carbon, nitrogen, and hydrogen atoms with (13)C, (15)N, and (2)H and observe the catalytic effects in HIV-1 protease. According to the Born-Oppenheimer approximation, isotopic substitution alters vibrational frequency with unchanged electrostatic properties. With the use of a fluorescent peptide to report on multiple steps in the reaction, we observe significantly reduced rates in the heavy enzyme relative to the light enzyme. A possible interpretation of our results is that there exists a dynamic link between mass-dependent bond vibrations of the enzyme and events in the reaction coordinate., (© 2011 American Chemical Society)

Human 5'-methylthioadenosine phosphorylase (MTAP) links the polyamine biosynthetic and S-adenosyl-l-methionine salvage pathways and is a target for anticancer drugs. p-Cl-PhT-DADMe-ImmA is a 10 pM, slow-onset tight-binding transition state analogue inhibitor of the enzyme. Titration of homotrimeric MTAP with this inhibitor established equivalent binding and independent catalytic function of the three catalytic sites. Thermodynamic analysis of MTAP with tight-binding inhibitors revealed entropic-driven interactions with small enthalpic penalties. A large negative heat capacity change of -600 cal/(mol K) upon inhibitor binding to MTAP is consistent with altered hydrophobic interactions and release of water. Crystal structures of apo MTAP and MTAP in complex with p-Cl-PhT-DADMe-ImmA were determined at 1.9 and 2.0 Å resolution, respectively. Inhibitor binding caused condensation of the enzyme active site, reorganization at the trimer interfaces, the release of water from the active sites and subunit interfaces, and compaction of the trimeric structure. These structural changes cause the entropy-favored binding of transition state analogues. Homotrimeric human MTAP is contrasted to the structurally related homotrimeric human purine nucleoside phosphorylase. p-Cl-PhT-DADMe-ImmA binding to MTAP involves a favorable entropy term of -17.6 kcal/mol with unfavorable enthalpy of 2.6 kcal/mol. In contrast, binding of an 8.5 pM transition state analogue to human PNP has been shown to exhibit the opposite behavior, with an unfavorable entropy term of 3.5 kcal/mol and a favorable enthalpy of -18.6 kcal/mol. Transition state analogue interactions reflect protein architecture near the transition state, and the profound thermodynamic differences for MTAP and PNP suggest dramatic differences in contributions to catalysis from protein architecture.

Contributions of fast (femtosecond) dynamic motion to barrier crossing at enzyme catalytic sites is in dispute. Human purine nucleoside phosphorylase (PNP) forms a ribocation-like transition state in the phosphorolysis of purine nucleosides and fast protein motions have been proposed to participate in barrier crossing. In the present study, (13)C-, (15)N-, (2)H-labeled human PNP (heavy PNP) was expressed, purified to homogeneity, and shown to exhibit a 9.9% increase in molecular mass relative to its unlabeled counterpart (light PNP). Kinetic isotope effects and steady-state kinetic parameters were indistinguishable for both enzymes, indicating that transition-state structure, equilibrium binding steps, and the rate of product release were not affected by increased protein mass. Single-turnover rate constants were slowed for heavy PNP, demonstrating reduced probability of chemical barrier crossing from enzyme-bound substrates to enzyme-bound products. In a second, independent method to probe barrier crossing, heavy PNP exhibited decreased forward commitment factors, also revealing mass-dependent decreased probability for barrier crossing. Increased atomic mass in human PNP alters bond vibrational modes on the femtosecond time scale and reduces on-enzyme chemical barrier crossing. This study demonstrates coupling of enzymatic bond vibrations on the femtosecond time scale to barrier crossing.

The reversible phosphorolysis of uridine to generate uracil and ribose 1-phosphate is catalyzed by uridine phosphorylase and is involved in the pyrimidine salvage pathway. We define the reaction mechanism of uridine phosphorylase from Trypanosoma cruzi by steady-state and pre-steady-state kinetics, pH-rate profiles, kinetic isotope effects from uridine, and solvent deuterium isotope effects. Initial rate and product inhibition patterns suggest a steady-state random kinetic mechanism. Pre-steady-state kinetics indicated no rate-limiting step after formation of the enzyme-products ternary complex, as no burst in product formation is observed. The limiting single-turnover rate constant equals the steady-state turnover number; thus, chemistry is partially or fully rate limiting. Kinetic isotope effects with [1'-(3)H]-, [1'-(14)C]-, and [5'-(14)C,1,3-(15)N(2)]uridine gave experimental values of (α-T)(V/K)(uridine) = 1.063, (14)(V/K)(uridine) = 1.069, and (15,β-15)(V/K)(uridine) = 1.018, in agreement with an A(N)D(N) (S(N)2) mechanism where chemistry contributes significantly to the overall rate-limiting step of the reaction. Density functional theory modeling of the reaction in gas phase supports an A(N)D(N) mechanism. Solvent deuterium kinetic isotope effects were unity, indicating that no kinetically significant proton transfer step is involved at the transition state. In this N-ribosyl transferase, proton transfer to neutralize the leaving group is not part of transition state formation, consistent with an enzyme-stabilized anionic uracil as the leaving group. Kinetic analysis as a function of pH indicates one protonated group essential for catalysis and for substrate binding.

Uridine phosphorylase catalyzes the reversible phosphorolysis of uridine and 2'-deoxyuridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli , purified to homogeneity, and shown to be a homodimeric uridine phosphorylase, with similar specificity for uridine and 2'-deoxyuridine and undetectable activity toward thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1'-(14)C = 1.103, 1,3-(15)N(2) = 1.034, 3-(15)N = 1.004, 1-(15)N = 1.030, 1'-(3)H = 1.132, 2'-(2)H = 1.086, and 5'-(3)H(2) = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition-state structure and geometry. Matching of experimental KIEs to proposed transition-state structures suggests an almost synchronous, S(N)2-like transition-state model, in which the ribosyl moiety possesses significant bond order to both nucleophile and leaving groups. Natural bond orbital analysis allowed a comparison of the charge distribution pattern between the ground-state and the transition-state models.

DNA (cytosine-5)-methyltransferases (DNMTs) catalyze the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 5-position of cytosine residues and thereby silence transcription of regulated genes. DNMTs are important epigenetic targets. However, isolated DNMTs are weak catalysts and are difficult to assay. We report an ultrasensitive luciferase-linked continuous assay that converts the S-adenosyl-L-homocysteine product of DNA methylation to a quantifiable luminescent signal. Results with this assay are compared with the commonly used DNA labeling from [methyl-(3)H]AdoMet. A 5'-methylthioadenosine-adenosylhomocysteine nucleosidase is used to hydrolyze AdoHcy to adenine. Adenine phosphoribosyl transferase converts adenine to AMP and pyruvate orthophosphate dikinase converts AMP to ATP. Firefly luciferase gives a stable luminescent signal that results from continuous AMP recycling to ATP. This assay exhibits a broad dynamic range (0.1-1000 pmol of AdoHcy). The rapid response time permits continuous assays of DNA methylation detected by light output. The assay is suitable for high-throughput screening of chemical libraries for DNMT inhibition activity. The kinetic properties of human and bacterial CpG methyltransferases are characterized using this assay. Human catalytic domain DNMT3b activation by DNMT3L is shown to involve two distinct kinetic states that alter k(cat) but not K(m) for AdoMet. The assay is shown to be robust in the presence of high concentrations of the pyrimidine analogues 5-azacytidine and 5-azacytosine.

Orotate phosphoribosyltransferases (OPRTs) form and break the N-ribosidic bond to pyrimidines by way of ribocation-like transition states (TSs) and therefore exhibit large α-secondary 1'-(3)H k(cat)/K(m) kinetic isotope effects (KIEs) [Zhang, Y., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 8787-8794]. Substrate binding isotope effects (BIEs) with OPRTs report on the degree of ground-state destabilization for these complexes and permit resolution of binding and transition-state effects from the k(cat)/K(m) KIEs. The BIEs for interactions of [1'-(3)H]orotidine 5'-monophosphate (OMP) with the catalytic sites of Plasmodium falciparum and human OPRTs are 1.104 and 1.108, respectively. These large BIEs establish altered sp(3) bond hybridization of C1' toward the sp(2) geometry of the transition states upon OMP binding. Thus, the complexes of these OPRTs distort OMP part of the way toward the transition state. As the [1'-(3)H]OMP k(cat)/K(m) KIEs are approximately 1.20, half of the intrinsic k(cat)/K(m) KIEs originate from BIEs. Orotidine, a slow substrate for these enzymes, binds to the catalytic site with no significant [1'-(3)H]orotidine BIEs. Thus, OPRTs are unable to initiate ground-state destabilization of orotidine by altered C1' hybridization because of the missing 5'-phosphate. However the k(cat)/K(m) KIEs for [1'-(3)H]orotidine are also approximately 1.20. The C1' distortion for OMP happens in two steps, half upon binding and half on going from the Michaelis complex to the TS. With orotidine as the substrate, there is no ground-state destabilization in the Michaelis complexes, but the C1' distortion at the TS is equal to that of OMP. The large single barrier for TS formation with orotidine slows the rate of barrier crossing.

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of 6-oxypurine (2'-deoxy)ribonucleosides, generating (2-deoxy)ribose 1-phosphate and the purine base. Transition-state models for inosine cleavage have been proposed with bovine, human, and malarial PNPs using arsenate as the nucleophile, since kinetic isotope effects (KIEs) are obscured on phosphorolysis due to high commitment factors. The Phe200Gly mutant of human PNP has low forward and reverse commitment factors in the phosphorolytic reaction, permitting the measurement of competitive intrinsic KIEs on both arsenolysis and phosphorolysis of inosine. The intrinsic 1'-(14)C, 1'-(3)H, 2'-(2)H, 9-(15)N, and 5'-(3)H(2) KIEs for inosine were measured for arsenolysis and phosphorolysis. Except for the remote 5'-(3)H(2), and some slight difference between the 2'-(2)H KIEs, all isotope effects originating in the reaction coordinate are the same within experimental error. Hence, arsenolysis and phosphorolysis proceed through closely related transition states. Although electrostatically similar, the volume of arsenate is greater than phosphate and supports a steric influence to explain the differences in the 5'-(3)H(2) KIEs. Density functional theory calculations provide quantitative models of the transition states for Phe200Gly human PNP-catalyzed arsenolysis and phosphorolysis, selected upon matching calculated and experimental KIEs. The models confirm the striking resemblance between the transition states for the two reactions.

Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K(m) = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap(4)A (2.0 Å resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg(2+) ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer α/β/α sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.

Human thymidine phosphorylase (hTP) is responsible for thymidine (dT) homeostasis, promotes angiogenesis, and is involved in metabolic inactivation of antiproliferative agents that inhibit thymidylate synthase. Understanding its transition state structure is on the path to design transition state analogues. Arsenolysis of dT by hTP permits kinetic isotope effect (KIE) analysis of the reaction by forming thymine and the chemically unstable 2-deoxyribose 1-arsenate. The transition state for the arsenolytic reaction was characterized using multiple KIEs and computational analysis. Transition state analysis revealed a concerted bimolecular (A(N)D(N)) mechanism. A transition state constrained to match the intrinsic KIE values was found using density functional theory (B3LYP/6-31G*). An active site histidine is implicated as the catalytic base responsible for activation of the arsenate nucleophile and stabilization of the thymine leaving group during the isotopically sensitive step. At the transition state, the deoxyribose ring exhibits significant oxocarbenium ion character with bond breaking (r(C-N) = 2.45 Å) nearly complete and minimal bond making to the attacking nucleophile (r(C-O) = 2.95 Å). The transition state model predicts a deoxyribose conformation with a 2'-endo ring geometry. Transition state structure for the slow hydrolytic reaction of hTP involves a stepwise mechanism [Schwartz, P. A., Vetticatt, M. J., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 13425-13433], in contrast to the concerted mechanism described here for arsenolysis.

The PA3004 gene of Pseudomonas aeruginosa PAO1 was originally annotated as a 5'-methylthioadenosine phosphorylase (MTAP). However, the PA3004 encoded protein uses 5'-methylthioinosine (MTI) as a preferred substrate and represents the only known example of a specific MTI phosphorylase (MTIP). MTIP does not utilize 5'-methylthioadenosine (MTA). Inosine is a weak substrate with a k(cat)/K(m) value 290-fold less than MTI and is the second best substrate identified. The crystal structure of P. aeruginosa MTIP (PaMTIP) in complex with hypoxanthine was determined to 2.8 Å resolution and revealed a 3-fold symmetric homotrimer. The methylthioribose and phosphate binding regions of PaMTIP are similar to MTAPs, and the purine binding region is similar to that of purine nucleoside phosphorylases (PNPs). The catabolism of MTA in P. aeruginosa involves deamination to MTI and phosphorolysis to hypoxanthine (MTA → MTI → hypoxanthine). This pathway also exists in Plasmodium falciparum, where the purine nucleoside phosphorylase (PfPNP) acts on both inosine and MTI. Three tight-binding transition state analogue inhibitors of PaMTIP are identified with dissociation constants in the picomolar range. Inhibitor specificity suggests an early dissociative transition state for PaMTIP. Quorum sensing molecules are associated with MTA metabolism in bacterial pathogens suggesting PaMTIP as a potential therapeutic target.

The S-adenosylmethionine (AdoMet) salvage enzyme 5'-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5'-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2(-/-)γC(-/-) and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP(-/-) and H358 MTAP(+/+) tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥ 3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy.

Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent.

Malaria is a leading cause of morbidity and mortality in the tropics. Chemotherapeutic and vector control strategies have been applied for more than a century but have not been efficient in disease eradication. Increased resistance of malaria parasites to drug treatment and of mosquito vectors to insecticides requires the development of novel chemotherapeutic agents. Malaria parasites exhibit rapid nucleic acid synthesis during their intraerythrocytic growth phase. Plasmodium purine and pyrimidine metabolic pathways are distinct from those of their human hosts. Thus, targeting purine and pyrimidine metabolic pathways provides a promising route for novel drug development. Recent developments in enzymatic transition state analysis have provided an improved route to inhibitor design targeted to specific enzymes, including those of purine and pyrimidine metabolism. Modern transition state analogue drug discovery has resulted in transition state analogues capable of binding to target enzymes with unprecedented affinity and specificity. These agents can provide specific blocks in essential pathways. The combination of tight binding with the high specificity of these logically designed inhibitors, results in low toxicity and minor side effects. These features reduce two of the major problems with the current antimalarials. Transition state analogue design is being applied to generate new lead compounds to treat malaria by targeting purine and pyrimidine pathways.

Experimental analysis of enzymatic transition-state structures uses kinetic isotope effects (KIEs) to report on bonding and geometry differences between reactants and the transition state. Computational correlation of experimental values with chemical models permits three-dimensional geometric and electrostatic assignment of transition states formed at enzymatic catalytic sites. The combination of experimental and computational access to transition-state information permits (a) the design of transition-state analogs as powerful enzymatic inhibitors, (b) exploration of protein features linked to transition-state structure, (c) analysis of ensemble atomic motions involved in achieving the transition state, (d) transition-state lifetimes, and (e) separation of ground-state (Michaelis complexes) from transition-state effects. Transition-state analogs with picomolar dissociation constants have been achieved for several enzymatic targets. Transition states of closely related isozymes indicate that the protein's dynamic architecture is linked to transition-state structure. Fast dynamic motions in catalytic sites are linked to transition-state generation. Enzymatic transition states have lifetimes of femtoseconds, the lifetime of bond vibrations. Binding isotope effects (BIEs) reveal relative reactant and transition-state analog binding distortion for comparison with actual transition states.

Dynamic motions of human purine nucleoside phosphorylase (hPNP) in complex with transition-state analogues and reactants were studied using 10 ns explicit solvent molecular dynamics simulations. hPNP is a homotrimer that catalyzes the phosphorolysis of purine 6-oxynucleosides. The ternary complex of hPNP includes the binding of a ligand and phosphate to the active site. Molecular dynamics simulations were performed on the ternary complex of six ligands including the picomolar transition-state analogues, Immucillin-H (K(d) = 56 pM), DADMe-Immucillin-H (K(d) = 8.5 pM), DATMe-Immucillin-H (K(d) = 8.6 pM), SerMe-Immucillin-H (K(d) = 5.2 pM), the substrate inosine, and a complex containing only phosphate. Protein-inhibitor complexes of the late transition-state inhibitors, DADMe-Imm-H and DATMe-Imm-H, are inflexible. Despite the structural similarity of SerMe-Imm-H and DATMe-Imm-H, the protein complex of SerMe-Imm-H is flexible, and the inhibitor is highly mobile within the active sites. All inhibitors exhibit an increased number of nonbonding interactions in the active site relative to the substrate inosine. Water density within the catalytic site is lower for DADMe-ImmH, DATMe-Imm-H, and SerMe-Imm-H than that for the substrate inosine. Tight binding of the picomolar inhibitors results from increased interactions within the active site and a reduction in the number of water molecules organized within the catalytic site relative to the substrate inosine.

Plasmodium falciparum orotate phosphoribosyltransferase (PfOPRT) catalyzes the reversible pyrophosphorolysis of orotidine 5'-monophosphate (OMP). Transition-state analysis from kinetic isotope effects supports a dianionic orotic acid (OA) leaving group. Isotope-edited Fourier transform infrared (FTIR) spectrometry complemented by homology modeling and quantum chemical calculations were used to characterize the orotate hydrogen-bond network for PfOPRT. Bond stretch frequencies for C(2)═O and C(4)═O of OMP were established from (13)C-edited FTIR difference spectra. Both frequencies were shifted downward by 20 cm(-1) upon formation of the Michaelis complex. Hydrogen-bond interactions to the orotate moiety induce strong leaving group polarization by ground-state destabilization. The C(2)═O bond is 2.7 Å from two conserved water molecules, and the C(4)═O bond is within 2.4 Å of the NH(2)(ω) of Arg241 and the peptide NH of Phe97. Relative to free OMP, the N1 atom of PfOPRT-bound OMP indicates a ΔpK(a) of -4.6. The decreased basicity of N1 supports leaving group activation through a hydrogen-bond network at the PfOPRT active site. PfOPRT in complex with (18)O-PPi and a proposed transition-state analogue revealed a trianionic PPi nucleophile with no significant P··O bond polarization, supporting a mechanism proceeding through the migration of the ribocation toward the PPi. These results along with previous PfOPRT transition-state analyses provide reaction coordinate information for the PfOPRT-catalyzed OMP pyrophosphorolysis reaction.

Human thymidine phosphorylase (hTP) is responsible for thymidine (dT) homeostasis, and its action promotes angiogenesis. In the absence of phosphate, hTP catalyzes a slow hydrolytic depyrimidination of dT yielding thymine and 2-deoxyribose (dRib). Its transition state was characterized using multiple kinetic isotope effect (KIE) measurements. Isotopically enriched thymidines were synthesized enzymatically from glucose or (deoxy)ribose, and intrinsic KIEs were used to interpret the transition state structure. KIEs from [1'-(14)C]-, [1-(15)N]-, [1'-(3)H]-, [2'R-(3)H]-, [2'S-(3)H]-, [4'-(3)H]-, and [5'-(3)H]dTs provided values of 1.033 ± 0.002, 1.004 ± 0.002, 1.325 ± 0.003, 1.101 ± 0.004, 1.087 ± 0.005, 1.040 ± 0.003, and 1.033 ± 0.003, respectively. Transition state analysis revealed a stepwise mechanism with a 2-deoxyribocation formed early and a higher energetic barrier for nucleophilic attack of a water molecule on the high energy intermediate. An equilibrium exists between the deoxyribocation and reactants prior to the irreversible nucleophilic attack by water. The results establish activation of the thymine leaving group without requirement for phosphate. A transition state constrained to match the intrinsic KIEs was found using density functional theory. An active site histidine (His116) is implicated as the catalytic base for activation of the water nucleophile at the rate-limiting transition state. The distance between the water nucleophile and the anomeric carbon (r(C-O)) is predicted to be 2.3 A at the transition state. The transition state model predicts that deoxyribose adopts a mild 3'-endo conformation during nucleophilic capture. These results differ from the concerted bimolecular mechanism reported for the arsenolytic reaction (Birck, M. R.; Schramm, V. L. J. Am. Chem. Soc. 2004, 126, 2447-2453).

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a dual substrate bacterial enzyme involved in S-adenosylmethionine (SAM) related quorum sensing pathways that regulates virulence in many bacterial species. MTANs from many bacteria are directly involved in the quorum sensing mechanism by regulating the synthesis of autoinducer molecules that are used by bacterial communities to communicate. In humans, 5'-methylthioadenosine phosphorylase (MTAP) is involved in polyamine biosynthesis as well as in purine and SAM salvage pathways and thus has been identified as an anticancer target. Previously we have described the synthesis and biological activity of several aza-C-nucleoside mimics with a sulfur atom at the 5' position that are potent E. coli MTAN and human MTAP inhibitors. Because of the possibility that the sulfur may affect bioavailability, we were interested in synthesizing "sulfur-free" analogues. Herein we describe the preparation of a series of "sulfur-free" transition state analogue inhibitors of E. coli MTAN and human MTAP that have low nano- to picomolar dissociation constants and are potentially novel bacterial anti-infective and anticancer drug candidates.

2-Ethoxyethaneseleninic acid reacts with electron-rich aromatic substrates to deliver, by way of the selenoxides, the (2-ethoxyethyl)seleno ethers, which can in turn be transformed into a diverse set of aryl-selenylated products. Among these, a family of 5-uridinyl derivatives shows submicromolar inhibition of human and malarial orotate phosphoribosyltransferase.

Orotate phosphoribosyltransferases from Plasmodium falciparum and human sources (PfOPRT and HsOPRT) use orotidine as a slow substrate in the pyrophosphorolysis reaction. With orotidine, intrinsic kinetic isotope effects (KIEs) can be measured for pyrophosphorolysis, providing the first use of pyrophosphate (PPi) in solving an enzymatic transition state. Transition-state structures of PfOPRT and HsOPRT were solved through quantum chemical matching of computed and experimental intrinsic KIEs and can be compared to transition states solved with pyrophosphate analogues as slow substrates. PfOPRT and HsOPRT are characterized by late transition states with fully dissociated orotate, well-developed ribocations, and weakly bonded PPi nucleophiles. The leaving orotates are 2.8 A distant from the anomeric carbons at the transition states. Weak participation of the PPi nucleophiles gives C1'-O(PPi) bond distances of approximately 2.3 A. These transition states are characterized by C2'-endo ribosyl pucker, based on the beta-secondary [2'-(3)H] KIEs. The geometry at the 5'-region is similar for both enzymes, with C3'-C4'-C5'-O5' dihedral angles near -170 degrees . These novel phosphoribosyltransferase transition states are similar to but occur earlier in the reaction coordinate than those previously determined with orotidine 5'-monophosphate and phosphonoacetic acid as substrates. The similarity between the transition states with different substrate analogues supports similar transition state structures imposed by PfOPRT and HsOPRT even with distinct reactants. We propose that the transition state similarity with different nucleophiles is determined, in part, by the geometric constraints imposed by the catalytic sites.

Isotope-edited difference Raman and FTIR studies complemented by ab initio calculations have been applied to the transition state analogue complex of HGPRT.ImmHP.MgPP(i) to determine the ionic states of the 5'-phosphate moiety of ImmHP and of PP(i). These measurements characterize electrostatic interactions within the enzyme active site as deduced from frequency shifts of the phosphate groups. The bound 5'-phosphate moiety of ImmHP is dianionic, and this phosphate group exists in two different conformations within the protein complex. In one conformation, a hydrogen bond between the 5'-phosphate of ImmHP and the OH group of Tyr104 in the catalytic loop appears to be stronger. With the stronger H-bond, the OH of Tyr104 approaches one of the P..O bonds from the bridging oxygen side to cause distortion of the PO(3) moiety, as indicated by a lowered symmetric P..O stretch frequency. The asymmetric stretch frequencies are similar in both phosphate conformations. Bound PP(i) in this complex is fully ionized to P(2)O(7)(4-). Bond frequency changes for bound PP(i) indicate coordination to Mg(2+) ions but show no indication of significant P..O bond polarization. Extrapolation of these results to reaction coordinate motion for HGPRT suggests that bond formation between C1' of the nucleotide ribose and the oxygen of PP(i) is accomplished by migration of the ribocation toward immobilized pyrophosphate.

Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (K*i(1/4) 58 pM, first generation)contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (K*i(1/4) 9 pM, second-generation),uses a methylene-bridged dihydroxypyrrolidine cation with twoasymmetric centers.DATMe-Immucillin-H (K*i(1/4)9 pM, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (K*i(1/4) 5 pM, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; 1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, 2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, 3) interaction between phosphate and inhibitor hydroxyl groups, and 4) His257 interacting with the 5'-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second- to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transition-state analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.

Human purine nucleoside phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; K(d) = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; K(d) = 8.6 pM). ImmH binds with a larger entropic penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP.PO(4).inosine <--> PNP.Hx.R-1-P) and inhibited complexes (PNP.PO(4).DATMe-ImmH and PNP.PO(4).ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP.PO(4)) in extended incubations. The PNP.PO(4).ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely with entropy of binding than with affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation.

Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the K(m) and/or K(i) for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the K(i) for PfENT1 transport of hypoxanthine and adenosine is in the 300-700microM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport K(m). PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [(3)H]hypoxanthine or [(3)H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.

Ricin A-chain (RTA) and saporin-L1 (SAP) catalyze adenosine depurination of 28S rRNA to inhibit protein synthesis and cause cell death. We present the crystal structures of RTA and SAP in complex with transition state analogue inhibitors. These tight-binding inhibitors mimic the sarcin-ricin recognition loop of 28S rRNA and the dissociative ribocation transition state established for RTA catalysis. RTA and SAP share unique purine-binding geometry with quadruple pi-stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the pi-stack provides cationic polarization and enhanced leaving group ability to the susceptible adenine. Common features of these ribosome-inactivating proteins include adenine leaving group activation, a remarkable lack of ribocation stabilization, and conserved glutamates as general bases for activation of the H(2)O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.

Ribosome inactivating proteins (RIPs) catalyze the hydrolytic depurination of one or more adenosine residues from eukaryotic ribosomes. Depurination of the ribosomal sarcin-ricin tetraloop (GAGA) causes inhibition of protein synthesis and cellular death. We characterized the catalytic properties of saporin-L1 from Saponaria officinalis (soapwort) leaves, and it demonstrated robust activity against defined nucleic acid substrates and mammalian ribosomes. Transition state analogue mimics of small oligonucleotide substrates of saporin-L1 are powerful, slow-onset inhibitors when adenosine is replaced with the transition state mimic 9-deazaadenine-9-methylene-N-hydroxypyrrolidine (DADMeA). Linear, cyclic, and stem-loop oligonucleotide inhibitors containing DADMeA and based on the GAGA sarcin-ricin tetraloop gave slow-onset tight-binding inhibition constants (K(i)*) of 2.3-8.7 nM under physiological conditions and bind up to 40000-fold tighter than RNA substrates. Saporin-L1 inhibition of rabbit reticulocyte translation was protected by these inhibitors. Transition state analogues of saporin-L1 have potential in cancer therapy that employs saporin-L1-linked immunotoxins.

Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA [Tyler, P. C., Taylor, E. A., Frohlich, R. G. G., and Schramm, V. L. (2007) J. Am. Chem. Soc. 129, 6872-6879]. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation [Larson, E. T., et al. (2008) J. Mol. Biol. 381, 975-988]. Here, the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin reveals an unprecedented binding geometry for 5'-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5'-methylthioribosyl groups are rotated 130 degrees . A hydrogen bonding network between Asp172 and the 3'-hydroxyl of MT-coformycin is essential for recognition of the 5'-methylthioribosyl group. Water occupies the 5'-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to the corresponding purine base and alpha-D-ribose 1-phosphate. Its genetic loss causes a lethal T cell deficiency. The highly reactive ribocation transition state of human PNP is protected from solvent by hydrophobic residues that sequester the catalytic site. The catalytic site was enlarged by replacing individual catalytic site amino acids with glycine. Reactivity of the ribocation transition state was tested for capture by water and other nucleophiles. In the absence of phosphate, inosine is hydrolyzed by native, Y88G, F159G, H257G, and F200G enzymes. Phosphorolysis but not hydrolysis is detected when phosphate is bound. An unprecedented N9-to-N3 isomerization of inosine is catalyzed by H257G and F200G in the presence of phosphate and by all PNPs in the absence of phosphate. These results establish a ribocation lifetime too short to permit capture by water. An enlarged catalytic site permits ribocation formation with relaxed geometric constraints, permitting nucleophilic rebound and N3-inosine isomerization.

Nicotinamide phosphoribosyltransferase (NAMPT) is highly evolved to capture nicotinamide (NAM) and replenish the nicotinamide adenine dinucleotide (NAD(+)) pool during ADP-ribosylation and transferase reactions. ATP-phosphorylation of an active-site histidine causes catalytic activation, increasing NAM affinity by 160,000. Crystal structures of NAMPT with catalytic site ligands identify the phosphorylation site, establish its role in catalysis, demonstrate unique overlapping ATP and phosphoribosyltransferase sites, and establish reaction coordinate motion. NAMPT structures with beryllium fluoride indicate a covalent H247-BeF(3)(-) as the phosphohistidine mimic. Activation of NAMPT by H247-phosphorylation causes stabilization of the enzyme-phosphoribosylpyrophosphate complex, permitting efficient capture of NAM. Reactant and product structures establish reaction coordinate motion for NAMPT to be migration of the ribosyl anomeric carbon from the pyrophosphate leaving group to the nicotinamide-N1 while the 5-phosphoryl group, the pyrophosphate moiety, and the nicotinamide ring remain fixed in the catalytic site.

What are the atomic motions at enzymatic catalytic sites on the timescale of chemical change? Combined experimental and computational chemistry approaches take advantage of transition-state analogs to reveal dynamic motions linked to transition-state formation. QM/MM transition path sampling from reactive complexes provides both temporal and dynamic information for barrier crossing. Fast (femtosecond to picosecond) dynamic motions provide essential links to enzymatic barrier crossing by local or promoting-mode dynamic searches through bond-vibrational space. Transition-state lifetimes are within the femtosecond timescales of bond vibrations and show no manifestations of stabilized, equilibrated complexes. The slow binding and protein conformational changes (microsecond to millisecond) also required for catalysis are temporally decoupled from the fast dynamic motions forming the transition state. According to this view of enzymatic catalysis, transition states are formed by fast, coincident dynamic excursions of catalytic site elements, while the binding of transition-state analogs is the conversion of the dynamic excursions to equilibrated states.

Human purine nucleoside phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency, and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occurs upon binding to the first of three subunits, and binding to subsequent sites occurs with negative cooperativity. In contrast, substrate analogue and product bind without cooperativity. Titrations of human PNP using isothermal calorimetry indicate that binding of a structurally rigid first-generation Immucillin (K(d) = 56 pM) is driven by large negative enthalpy values (DeltaH = -21.2 kcal/mol) with a substantial entropic (-TDeltaS) penalty. The tightest-binding inhibitors (K(d) = 5-9 pM) have increased conformational flexibility. Despite their conformational freedom in solution, flexible inhibitors bind with high affinity because of reduced entropic penalties. Entropic penalties are proposed to arise from conformational freezing of the PNP.inhibitor complex with the entropy term dominated by protein dynamics. The conformationally flexible Immucillins reduce the system entropic penalty. Disrupting the ribosyl 5'-hydroxyl interaction of transition state analogues with PNP causes favorable entropy of binding. Tight binding of the 17 Immucillins is characterized by large enthalpic contributions, emphasizing their similarity to the transition state. Via introduction of flexibility into the inhibitor structure, the enthalpy-entropy compensation pattern is altered to permit tighter binding.

Human PNP is a homotrimer containing three tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The catalytic sites of PNP are located near the subunit-subunit interfaces where F159 is a catalytic site residue donated from an adjacent subunit. F159 covers the top (beta) surface of the ribosyl group at the catalytic site. QM/MM calculations of human PNP have shown that F159 is the center of the most mobile region of the protein providing access to the substrate in the active site. F159 is also the key residue in a cluster of hydrophobic residues that shield catalytic site ligands from bulk solvent. Trp-free human PNP (Leuko-PNP) was previously engineered by replacing the three Trp residues of native PNP with Tyr. From this active construct, a single Trp residue was placed in the catalytic site loop (F159W-Leuko-PNP) as a reporter group for the ribosyl region of the catalytic site. The F159W-Leuko-PNP fluorescence is red shifted compared to native PNP, suggesting a solvent-exposed Trp residue. Upon ligand binding (hypoxanthine), the 3-fold fluorescence quench confirms conformational packing of the catalytic site pocket hydrophobic cluster. F159W-Leuko-PNP has an on-enzyme thermodynamic equilibrium constant (Keq) near unity in the temperature range between 20 and 30 degrees C and nonzero enthalpic components, making it suitable for laser-induced T-jump analyses. T-jump relaxation kinetics of F159W-Leuko-PNP in equilibrium with substrates and/or products indicate the conformational equilibria of at least two ternary complex intermediates in the nano- to millisecond time scale (1000-10000 s-1) that equilibrate prior to the slower chemical step (approximately 200 s-1). F159W-Leuko-PNP provides a novel protein platform to investigate the protein conformational dynamics occurring prior to transition state formation.

Ricin is a family member of the lethal ribosome-inactivating proteins (RIP) found in plants. Ricin toxin A-chain (RTA) from castor beans catalyzes the hydrolytic depurination of a single base from a GAGA tetraloop of eukaryotic rRNA to release a single adenine from the sarcin-ricin loop (SRL). Protein synthesis is inhibited by loss of the elongation factor binding site resulting in cell death. We report a sensitive coupled assay for the measurement of adenine released from ribosomes or small stem-loop RNAs by RTA catalysis. Adenine phosphoribosyl transferase (APRTase) and pyruvate orthophosphate dikinase (PPDK) convert adenine to ATP for quantitation by firefly luciferase. The resulting AMP is cycled to ATP to give sustained luminescence proportional to adenine concentration. Subpicomole adenine quantitation permits the action of RTA on eukaryotic ribosomes to be followed in continuous, high-throughput assays. Facile analysis of RIP catalytic activity will have applications in plant toxin detection, inhibitor screens, mechanistic analysis of depurinating agents on oligonucleotides and intact ribosomes, and in cancer immunochemotherapy. Kinetic analysis of the catalytic action of RTA on rabbit reticulocyte 80S ribosomes establishes a catalytic efficiency of 2.6 x 10(8) M(-1) s(-1), a diffusion limited reaction indicating catalytic perfection even with large reactants.

Orotate phosphoribosyltransferases (OPRT) catalyze the formation of orotidine 5'-monophosphate (OMP) from alpha-D-phosphoribosylpyrophosphate (PRPP) and orotate, an essential step in the de novo biosynthesis of pyrimidines. Pyrimidine de novo biosynthesis is required in Plasmodium falciparum , and thus OPRT of the parasite (PfOPRT) is a target for antimalarial drugs. De novo biosynthesis of pyrimidines is also a feature of rapidly proliferating cancer cells. Human OPRT (HsOPRT) is therefore a target for neoplastic and autoimmune diseases. One approach to the inhibition of OPRTs is through analogues that mimic the transition states of PfOPRT and HsOPRT. The transition state structures of these OPRTs were analyzed by kinetic isotope effects (KIEs), substrate specificity, and computational chemistry. With phosphonoacetic acid (PA), an analogue of pyrophosphate, the intrinsic KIEs of [1'-(14)C], [1, 3-(15)N(2)], [3-(15)N], [1'-(3)H], [2'-(3)H], [4'-(3)H], and [5'-(3)H(2)] are 1.034, 1.028, 0.997, 1.261, 1.116, 0.974, and 1.013 for PfOPRT and 1.035, 1.025, 0.993, 1.199, 1.129, 0.962, and 1.019 for HsOPRT, respectively. Transition state structures of PfOPRT and HsOPRT were determined computationally by matching the calculated and intrinsic KIEs. The enzymes form late associative D(N)*A(N)(double dagger) transition states with complete orotate loss and partially associative nucleophile. The C1'-O(PA) distances are approximately 2.1 A at these transition states. The modest [1'-(14)C] KIEs and large [1'-(3)H] KIEs are characteristic of D(N)*A(N)(double dagger) transition states. The large [2'-(3)H] KIEs indicate a ribosyl 2'-C-endo conformation at the transition states. p-Nitrophenyl beta-D-ribose 5'-phosphate is a poor substrate of PfOPRT and HsOPRT but is a nanomolar inhibitor, supporting a reaction coordinate with strong leaving group activation.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a bacterial enzyme involved in S-adenosylmethionine-related quorum sensing pathways that induce bacterial pathogenesis factors. Transition state analogs MT-DADMe-Immucillin-A, EtT-DADMe-Immucillin-A and BuT-DADMe-Immucillin-A are slow-onset, tight-binding inhibitors of Vibrio cholerae MTAN (VcMTAN), with equilibrium dissociation constants of 73, 70 and 208 pM, respectively. Structural analysis of VcMTAN with BuT-DADMe-Immucillin-A revealed interactions contributing to the high affinity. We found that in V. cholerae cells, these compounds are potent MTAN inhibitors with IC(50) values of 27, 31 and 6 nM for MT-, EtT- and BuT-DADMe-Immucillin-A, respectively; the compounds disrupt autoinducer production in a dose-dependent manner without affecting growth. MT- and BuT-DADMe-Immucillin-A also inhibited autoinducer-2 production in enterohemorrhagic Escherichia coli O157:H7 with IC(50) values of 600 and 125 nM, respectively. BuT-DADMe-Immucillin-A inhibition of autoinducer-2 production in both strains persisted for several generations and caused reduction in biofilm formation. These results support MTAN's role in quorum sensing and its potential as a target for bacterial anti-infective drug design.

ImmH (1) and DADMe-ImmH (2) are potent inhibitors of human purine nucleoside phoshorylase (PNP), developed by us and currently in clinical trials for the treatment of a variety of T-cell related diseases. Compounds 1 and 2 were used as templates for the design and synthesis of a series of acyclic immucillin analogues (8-38) in order to identify simplified alternatives to 1 and 2. SerMe-ImmG (8) and DATMe-ImmG (9) displayed the lowest inhibition constants of 2.1 and 3.4 pM, respectively, vs PNP. It was postulated that the flexible natures of 8 and 9 enabled them to adopt conformations resembling those of 1 and 2 within the active site of PNP and that the positioning of two hydroxyl groups was critical for picomolar activity. SerMe-ImmH (10, K(d) = 5.2 pM) was shown to be orally available in mice with a long biological residence time on blood PNP.

Human malaria infections resulting from Plasmodium falciparum have become increasingly difficult to treat due to the emergence of drug-resistant parasites. The P. falciparum purine salvage enzyme purine nucleoside phosphorylase (PfPNP) is a potential drug target. Previous studies, in which PfPNP was targeted by transition state analogue inhibitors, found that those inhibiting human PNP and PfPNPs killed P. falciparum in vitro. However, many drugs have off-target interactions, and genetic evidence is required to demonstrate single target action for this class of potential drugs. We used targeted gene disruption in P. falciparum strain 3D7 to ablate PNP expression, yielding transgenic 3D7 parasites (Deltapfpnp). Lysates of the Deltapfpnp parasites showed no PNP activity, but activity of another purine salvage enzyme, adenosine deaminase (PfADA), was normal. When compared with wild-type 3D7, the Deltapfpnp parasites showed a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. Drug assays using immucillins, specific transition state inhibitors of PNP, were performed on wild-type and Deltapfpnp parasites. The Deltapfpnp parasites were more sensitive to PNP inhibitors that bound hPNP tighter and less sensitive to MT-ImmH, an inhibitor with 100-fold preference for PfPNP over hPNP. The results demonstrate the importance of purine salvage in P. falciparum and validate PfPNP as the target of immucillins.

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

Neighboring-group participation in the reaction catalyzed by purine nucleoside phosphorylase involves a compression mode between the 5'- and 4'-ribosyl oxygens, facilitated by His257. The His257Gly mutant opens a space in the catalytic site. Hydrophobic 5'-substituted Immucillins are transition-state analogue inhibitors of this mutant enzyme. Dissociation constants as low as 2pM are achieved, with K(m)/K(d) as high as 400,000,000.

Transition path sampling (TPS) has been applied to the chemical step of human purine nucleoside phosphorylase (PNP). The transition path ensemble provides insight into the detailed mechanistic dynamics and atomic motion involved in transition state passage. The reaction mechanism involves early loss of the ribosidic bond to form a transition state with substantial ribooxacarbenium ion character, followed by dynamic motion from the enzyme and a conformational change in the ribosyl group leading to migration of the anomeric carbon toward phosphate, to form the product ribose 1-phosphate. Calculations of the commitment probability along reactive paths demonstrated the presence of a broad energy barrier at the transition state. TPS identified (i) compression of the O4'...O5' vibrational motion, (ii) optimized leaving group interactions, and (iii) activation of the phosphate nucleophile as the reaction proceeds through the transition state region. Dynamic motions on the femtosecond timescale provide the simultaneous optimization of these effects and coincide with transition state formation.

Human nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) catalyzes the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PP i) from nicotinamide (NAM) and alpha- d-5-phosphoribosyl-1-pyrophosphate (PRPP). NAMPT, by capturing the energy provided by its facultative ATPase activity, allows the production of NMN at product:substrate ratios thermodynamically forbidden in the absence of ATP. With ATP hydrolysis coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, substrate affinity dramatically increases ( K m (NAM) from 855 to 5 nM), and the K eq shifts -2.1 kcal/mol toward NMN formation. ADP-ATP isotopic exchange experiments support the formation of a high-energy phosphorylated intermediate (phospho-H247) as the mechanism for altered catalytic efficiency during ATP hydrolysis. NAMPT captures only a small portion of the energy generated by ATP hydrolysis to shift the dynamic chemical equilibrium. Although the weak energetic coupling of ATP hydrolysis appears to be a nonoptimized enzymatic function, closer analysis of this remarkable protein reveals an enzyme designed to capture NAM with high efficiency at the expense of ATP hydrolysis. NMN is a rate-limiting precursor for recycling to the essential regulatory cofactor, nicotinamide adenine dinucleotide (NAD (+)). NMN synthesis by NAMPT is powerfully inhibited by both NAD (+) ( K i = 0.14 muM) and NADH ( K i = 0.22 muM), an apparent regulatory feedback mechanism.

The potent immucillin purine nucleoside phosphorylase (PNP ) inhibitors F-DADMe-ImmH [(3S,4S)-3], and [(3R,4R)-3] are synthesized in seven steps. Cycloaddition to a fluoroalkene and an enzymic resolution are the key features of the construction of the fluoropyrrolidines 11, from which the immucillins are assembled by use of a three-component Mannich reaction. Slow-onset binding constants (Ki(*)) for [(3S,4S)-3] and [(3R,4R)-3] with human PNP are 0.032 and 1.82 nM, respectively. F-DADMe-ImmH [(3S,4S)-3] exhibits oral availability in mice at doses as low as 0.2 mg/kg.

Sirtuin enzymes comprise a unique class of NAD(+)-dependent protein deacetylases. Although structures of many sirtuin complexes have been determined, structural resolution of intermediate chemical steps are needed to understand the deacetylation mechanism. We report crystal structures of the bacterial sirtuin, Sir2Tm, in complex with an S-alkylamidate intermediate, analogous to the naturally occurring O-alkylamidate intermediate, and a Sir2Tm ternary complex containing a dissociated NAD(+) analog and acetylated peptide. The structures and biochemical studies reveal critical roles for the invariant active site histidine in positioning the reaction intermediate, and for a conserved phenylalanine residue in shielding reaction intermediates from base exchange with nicotinamide. The new structural and biochemical studies provide key mechanistic insight into intermediate steps of the Sir2 deacetylation reaction.

Synthesis of [3'-2H]-labeled 5'-methylthioadenosine (MTA) derivatives permitted measurement of the [3'-2H] KIE of the reaction catalyzed by Streptococcus pneumoniae methylthioadenosine nucleosidase (spMTAN). The key [3'-2H] KIE revealed the partial 3'-OH polarization and H3'-endo-->exo ribosyl configuration at the spMTAN transition state. A new understanding of the transition state stabilization of spMTAN-catalyzed hydrolysis is uncovered in structural features at the spMTAN transition state.

Immucillin-H (BCX-1777, forodesine) is a transition state analogue and potent inhibitor of PNP that shows promise as a specific agent against activated human T-cells and T-cell leukemias. The immunosuppressive or antileukemic effects of Immucillin-H (ImmH) in cultured cells require co-administration with deoxyguanosine (dGuo) to attain therapeutic levels of intracellular dGTP. In this study we investigated the requirements for sensitivity and resistance to ImmH and dGuo. (3)H-ImmH transport assays demonstrated that the equilibrative nucleoside transporters (ENT1 and ENT2) facilitated the uptake of ImmH in human leukemia CCRF-CEM cells whereas (3)H-dGuo uptake was primarily dependent upon concentrative nucleoside transporters (CNTs). Analysis of lysates from ImmH-resistant CCRF-CEM-AraC-8D cells demonstrated undetectable deoxycytidine kinase (dCK) activity, suggesting that dCK and not deoxyguanosine kinase (dGK) was the rate-limiting enzyme for phosphorylation of dGuo in these cells. Examination of ImmH cytotoxicity in a hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient cell line CCRF-CEM-AraC-8C, demonstrated enhanced sensitivity to low concentrations of ImmH and dGuo. RT-PCR and sequencing of HGPRT from the HGPRT-deficient CCRF-CEM-AraC-8C cells identified an Exon 8 deletion mutation in this enzyme. Thus these studies show that specific nucleoside transporters are required for ImmH cytotoxicity and predict that ImmH may be more cytotoxic to 6-thioguanine (6-TG) or 6-thiopurine-resistant leukemia cells caused by HGPRT deficiency.