Picomolar transition state analogue inhibitors of human 5'-methylthioadenosine phosphorylase and X-ray structure with MT-immucillin-A

Methythioadenosine phosphorylase (MTAP) functions solely in the polyamine pathway of mammals to remove the methylthioadenosine (MTA) product from both spermidine synthase (2.5.1.16) and spermine synthase (2.5.1.22). Inhibition of polyamine synthesis is a validated anticancer target. We designed and synthesized chemically stable analogues for the proposed transition state of human MTAP on the basis of the known ribooxacarbenium character at all reported N-ribosyltransferase transition states [Schramm, V. L. (2003) Acc. Chem. Res. 36, 588-596]. Methylthio-immucillin-A (MT-ImmA) is an iminoribitol tight-binding transition state analogue inhibitor with an equilibrium dissociation constant of 1.0 nM. The immucillins resemble the ribooxacarbenium ion transition states of N-ribosyltransferases and are tightly bound as the N4' cations. An ion pair formed between the iminoribitol cation and phosphate anion mimics the ribooxacarbenium cation-phosphate anion pair formed at the transition state and is confirmed in the crystal structure. The X-ray crystal structure of human MTAP with bound MT-Imm-A also reveals that the 5'-methylthio group lies in a flexible hydrophobic pocket. Substitution of the 5'-methylthio group with a 5'-phenylthio group gives an equilibrium binding constant of 1.0 nM. Methylthio-DADMe-immucillin-A is a pyrrolidine analogue of the transition state with a methylene bridge between the 9-deazaadenine group and the pyrrolidine ribooxacarbenium mimic. It is a slow-onset inhibitor with a dissociation constant of 86 pM. Improved binding energy with DADMe-immucillin-A suggests that the transition state is more closely matched by increasing the distance between leaving group and ribooxacarbenium mimics, consistent with a more dissociative transition state. Increasing the hydrophobic volume near the 5'-position at the catalytic site with 5'-phenylthio-DADMe-immucillin-A gave a dissociation constant of 172 pM, slightly weaker than the 5'-methylthio group. p-Cl-phenylthio-DADMe-immucillin-A binds with a dissociation constant of 10 pM (K(m)/K(i) value of 500000), the tightest binding inhibitor reported for MTAP. These slow-onset, tight-binding transition state analogue inhibitors are the most powerful reported for MTAP and have sufficient affinity to be useful in inhibiting the polyamine pathway.     

Catalysis by entropic effects: the action of cytidine deaminase on 5,6-dihydrocytidine

In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.     

Achieving the ultimate physiological goal in transition state analogue inhibitors for purine nucleoside phosphorylase

Genetic deficiency of human purine nucleoside phosphorylase (PNP) causes T-cell immunodeficiency. The enzyme is therefore a target for autoimmunity disorders, tissue transplant rejection and T-cell malignancies. Transition state analysis of bovine PNP led to the development of immucillin-H (ImmH), a powerful inhibitor of bovine PNP but less effective for human PNP. The transition state of human PNP differs from that of the bovine enzyme and transition state analogues specific for the human enzyme were synthesized. Three first generation transition state analogues, ImmG (Kd = 42 pM), ImmH (Kd = 56 pM), and 8-aza-ImmH (Kd = 180 pM), are compared with three second generation DADMe compounds (4'-deaza-1'-aza-2'-deoxy-1'-(9-methylene)-immucillins) tailored to the transition state of human PNP. The second generation compounds, DADMe-ImmG (Kd = 7pM), DADMe-ImmH (Kd = 16 pM), and 8-aza-DADMe-ImmH (Kd = 2.0 nM), are superior for inhibition of human PNP by binding up to 6-fold tighter. The DADMe-immucillins are the most powerful PNP inhibitors yet described, with Km/Kd ratios up to 5,400,000. ImmH and DADMe-ImmH are orally available in mice; DADMe-ImmH is more efficient than ImmH. DADMe-ImmH achieves the ultimate goal in transition state inhibitor design in mice. A single oral dose causes inhibition of the target enzyme for the approximate lifetime of circulating erythrocytes.     

Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues

Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP x ImmA x PO4 and TvPNP x DADMe-ImmA x PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 A ionic interaction between a PO4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP x ImmA x PO4 structure at 3.5 A. However, the TvPNP x ImmA x PO4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP x DADMe-ImmA x PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP x ImmH x PO4, causing an unfavorable leaving-group interaction.     

Site-bound water and the shortcomings of a less than perfect transition state analogue

Kinetic measurements have shown that substantial enthalpy changes accompany substrate binding by cytidine deaminase, increasing markedly as the reaction proceeds from the ground state (1/K(m), DeltaH = -13 kcal/mol) to the transition state (1/K(tx), DeltaH = -20 kcal/mol) [Snider, M. J., et al. (2000) Biochemistry 39, 9746-9753]. In the present work, we determined the thermodynamic changes associated with the equilibrium binding of inhibitors by cytidine deaminase by isothermal titration calorimetry and van't Hoff analysis of the temperature dependence of their inhibition constants. The results indicate that the binding of the transition state analogue 3,4-dihydrouridine DeltaH = -21 kcal/mol), like that of the transition state itself (DeltaH = -20 kcal/mol), is associated with a large favorable change in enthalpy. The significantly smaller enthalpy change that accompanies the binding of 3,4-dihydrozebularine (DeltaH = -10 kcal/mol), an analogue of 3,4-dihydrouridine in which a hydrogen atom replaces this inhibitor's 4-OH group, is consistent with the view that polar interactions with the substrate at the site of its chemical transformation play a critical role in reducing the enthalpy of activation for substrate hydrolysis. The entropic shortcomings of 3,4-dihydrouridine, in capturing all of the free energy involved in binding the actual transition state, may arise from its inability to displace a water molecule that occupies the binding site normally occupied by product ammonia.     

Energetic mapping of transition state analogue interactions with human and Plasmodium falciparum purine nucleoside phosphorylases

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.     

Thermodynamic dissection of the binding energetics of KNI-272, a potent HIV-1 protease inhibitor

KNI-272 is a powerful HIV-1 protease inhibitor with a reported inhibition constant in the picomolar range. In this paper, a complete experimental dissection of the thermodynamic forces that define the binding affinity of this inhibitor to the wild-type and drug-resistant mutant V82F/184V is presented. Unlike other protease inhibitors, KNI-272 binds to the protease with a favorable binding enthalpy. The origin of the favorable binding enthalpy has been traced to the coupling of the binding reaction to the burial of six water molecules. These bound water molecules, previously identified by NMR studies, optimize the atomic packing at the inhibitor/protein interface enhancing van der Waals and other favorable interactions. These interactions offset the unfavorable enthalpy usually associated with the binding of hydrophobic molecules. The association constant to the drug resistant mutant is 100-500 times weaker. The decrease in binding affinity corresponds to an increase in the Gibbs energy of binding of 3-3.5 kcal/mol, which originates from less favorable enthalpy (1.7 kcal/mol more positive) and entropy changes. Calorimetric binding experiments performed as a function of pH and utilizing buffers with different ionization enthalpies have permitted the dissection of proton linkage effects. According to these experiments, the binding of the inhibitor is linked to the protonation/deprotonation of two groups. In the uncomplexed form these groups have pKs of 6.0 and 4.8, and become 6.6 and 2.9 in the complex. These groups have been identified as one of the aspartates in the catalytic aspartyl dyad in the protease and the isoquinoline nitrogen in the inhibitor molecule. The binding affinity is maximal between pH 5 and pH 6. At those pH values the affinity is close to 6 x 10(10) M(-1) (Kd = 16 pM). Global analysis of the data yield a buffer- and pH-independent binding enthalpy of -6.3 kcal/mol. Under conditions in which the exchange of protons is zero, the Gibbs energy of binding is -14.7 kcal/mol from which a binding entropy of 28 cal/K mol is obtained. Thus, the binding of KNI-272 is both enthalpically and entropically favorable. The structure-based thermodynamic analysis indicates that the allophenylnorstatine nucleus of KNI-272 provides an important scaffold for the design of inhibitors that are less susceptible to resistant mutations.     

Anopheles gambiae purine nucleoside phosphorylase: catalysis, structure, and inhibition

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 kcat 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 Km/Ki* of 5.4 x 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 A 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.PO4 complex than in HsPNP.DADMe-ImmH.SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.     

Purine nucleoside phosphorylase from Mycobacterium tuberculosis. Analysis of inhibition by a transition-state analogue and dissection by parts.

Purine salvage pathways are predicted to be present from the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis deoD gene encodes a presumptive purine nucleoside phosphorylase (PNP). The gene was cloned, expressed, purified, and found to exhibit PNP activity. Purified M. tuberculosis PNP is trimeric, similar to mammalian PNP's but unlike the hexameric Escherichia coli enzyme. Immucillin-H is a rationally designed analogue of the transition state that has been shown to be a potent inhibitor of mammalian PNP's. This inhibitor also exhibits slow-onset inhibition of M. tuberculosis PNP with a rapid, reversible inhibitor binding (K(i) of 2.2 nM) followed by an overall dissociation constant (K(i)) of 28 pM, yielding a K(m)/K(i) value of 10(6). Time-dependent tight binding of the inhibitor occurs with a rate of 0.1 s(-)(1), while relaxation of the complex is slower at 1.4 x 10(-)(3) s(-)(1). The pH dependence of the K(i) value of immucillin-H to the M. tuberculosis PNP suggests that the inhibitor binds as the neutral, unprotonated form that is subsequently protonated to generate the tight-binding species. The M. tuberculosis enzyme demonstrates independent and equivalent binding of immucilin-H at each of the three catalytic sites, unlike mammalian PNP. Analysis of the components of immucillin-H confirms that the inhibition gains most of its binding energy from the 9-deazahypoxanthine group (K(is) of 0.39 microM) while the 1,4-dideoxy-1,4-iminoribitol binds weakly (K(is) of 2.9 mM). Double-inhibition studies demonstrate antagonistic binding of 9-deazahypoxanthine and iminoribitol (beta = 13). However, the covalent attachment of these two components in immucillin-H increases equilibrium binding affinity by a factor of >14 000 (28 pM vs 0.39 microM) compared to 9-deazahypoxanthine alone, and by a factor of >10(8) compared to iminoribitol alone (28 pM vs 2.9 mM), from initial velocity measurements. The structural basis for M. tuberculosis PNP inhibition by immucillin-H and by its component parts is reported in the following paper [Shi, W., Basso, L. A., Santos, D. S., Tyler, P. C., Furneaux, R. H., Blanchard, J. S., Almo, S. C., and Schramm, V. L. (2001) Biochemistry 40, 8204-8215].     

Atomic dissection of the hydrogen bond network for transition-state analogue binding to purine nucleoside phosphorylase

Immucillin-H (ImmH) and immucillin-G (ImmG) were previously reported as transition-state analogues for bovine purine nucleoside phosphorylase (PNP) and are the most powerful inhibitors reported for the enzyme (K(i) = 23 and 30 pM). Sixteen new immucillins are used to probe the atomic interactions that cause tight binding for bovine PNP. Eight analogues of ImmH are identified with equilibrium dissociation constants of 1 nM or below. A novel crystal structure of bovine PNP-ImmG-PO(4) is described. Crystal structures of ImmH and ImmG bound to bovine PNP indicate that nearly every H-bond donor/acceptor site on the inhibitor is fully engaged in favorable H-bond partners. Chemical modification of the immucillins is used to quantitate the energetics for each contact at the catalytic site. Conversion of the 6-carbonyl oxygen to a 6-amino group (ImmH to ImmA) increases the dissociation constant from 23 pM to 2.6 million pM. Conversion of the 4'-imino group to a 4'-oxygen (ImmH to 9-deazainosine) increases the dissociation constant from 23 pM to 2.0 million pM. Substituents that induce small pK(a) changes at N-7 demonstrate modest loss of affinity. Thus, 8-F or 8-CH(3)-substitutions decrease affinity less than 10-fold. But a change in the deazapurine ring to convert N-7 from a H-bond donor to a H-bond acceptor (ImmH to 4-aza-3-deaza-ImmH) decreases affinity by >10(7). Introduction of a methylene bridge between 9-deazahypoxanthine and the iminoribitol (9-(1'-CH(2))-ImmH) increased the distance between leaving and oxacarbenium groups and increased K(i) to 91 000 pM. Catalytic site energetics for 20 substitutions in the transition-state analogue are analyzed in this approach. Disruption of the H-bond pattern that defines the transition-state ensemble leads to a large decrease in binding affinity. Changes in a single H-bond contact site cause up to 10.1 kcal/mol loss of binding energy, requiring a cooperative H-bond pattern in binding the transition-state analogues. Groups involved in leaving group activation and ribooxacarbenium ion stabilization are central to the H-bond network that provides transition-state stabilization and tight binding of the immucillins.     

Transition state structure of purine nucleoside phosphorylase and principles of atomic motion in enzymatic catalysis

Immucillin-H [ImmH; (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol] is a 23 pM inhibitor of bovine purine nucleoside phosphorylase (PNP) specifically designed as a transition state mimic [Miles, R. W., Tyler, P. C., Furneaux, R. H., Bagdassarian, C. K., and Schramm, V. L. (1998) Biochemistry 37, 8615-8621]. Cocrystals of PNP and the inhibitor are used to provide structural information for each step through the reaction coordinate of PNP. The X-ray crystal structure of free ImmH was solved at 0.9 A resolution, and a complex of PNP.ImmH.PO(4) was solved at 1.5 A resolution. These structures are compared to previously reported complexes of PNP with substrate and product analogues in the catalytic sites and with the experimentally determined transition state structure. Upon binding, ImmH is distorted to a conformation favoring ribosyl oxocarbenium ion formation. Ribosyl destabilization and transition state stabilization of the ribosyl oxocarbenium ion occur from neighboring group interactions with the phosphate anion and the 5'-hydroxyl of the ribosyl group. Leaving group activation of hypoxanthine involves hydrogen bonds to O6, N1, and N7 of the purine ring. Ordered water molecules provide a proton transfer bridge to O6 and N7 and permit reversible formation of these hydrogen bonds. Contacts between PNP and catalytic site ligands are shorter in the transition state analogue complex of PNP.ImmH.PO(4) than in the Michaelis complexes of PNP.inosine.SO(4) or PNP.hypoxanthine.ribose 1-PO(4). Reaction coordinate motion is dominated by translation of the carbon 1' of ribose between relatively fixed phosphate and purine groups. Purine and pyrimidine phosphoribosyltransferases and nucleoside N-ribosyl hydrolases appear to operate by a similar mechanism.     

Temperature effects on the catalytic efficiency, rate enhancement, and transition state affinity of cytidine deaminase, and the thermodynamic consequences for catalysis of removing a substrate "anchor"

To obtain a clearer understanding of the forces involved in transition state stabilization by Escherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substrate binding in the ground state and transition state for substrate hydrolysis. Viscosity studies indicate that the action of cytidine deaminase is not diffusion-limited. Thus, K(m) appears to be a true dissociation constant, and k(cat) describes the chemical reaction of the ES complex, not product release. Enzyme-substrate association is accompanied by a loss of entropy and a somewhat greater release of enthalpy. As the ES complex proceeds to the transition state (ES), there is little further change in entropy, but heat is taken up that almost matches the heat that was released with ES formation. As a result, k(cat)/K(m) (describing the overall conversion of the free substrate to ES is almost invariant with changing temperature. The free energy barrier for the enzyme-catalyzed reaction (k(cat)/K(m)) is much lower than that for the spontaneous reaction (k(non)) (DeltaDeltaG = -21.8 kcal/mol at 25 degrees C). This difference, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (S), is almost entirely enthalpic in origin (DeltaDeltaH = -20.2 kcal/mol), compatible with the formation of hydrogen bonds that stabilize the ES complex. Thus, the transition state affinity of cytidine deaminase increases rapidly with decreasing temperature. When a hydrogen bond between Glu-91 and the 3'-hydroxyl moiety of cytidine is disrupted by truncation of either group, k(cat)/K(m) and transition state affinity are each reduced by a factor of 10(4). This effect of mutation is entirely enthalpic in origin (DeltaDeltaH approximately 7.9 kcal/mol), somewhat offset by a favorable change in the entropy of transition state binding. This increase in entropy is attributed to a loss of constraints on the relative motions of the activated substrate within the ES complex. In an Appendix, some objections to the conventional scheme for transition state binding are discussed.     

Immucillin-H binding to purine nucleoside phosphorylase reduces dynamic solvent exchange

The rate and extent of hydrogen/deuterium (H/D) exchange into purine nucleoside phosphorylase (PNP) was monitored by electrospray ionization mass spectrometry (ESI-MS) to probe protein conformational and dynamic changes induced by a substrate analogue, products, and a transition state analogue. The genetic deficiency of PNP in humans is associated with severe T-cell immunodeficiency, while B-cell immunity remains functional. Inhibitors of PNP have been proposed for treatment of T-cell leukemia, to suppress the graft-vs.-host response, or to counter type IV autoimmune diseases without destroying humoral immunity. Calf spleen PNP is a homotrimer of polypeptide chains with 284 amino residues, molecular weight 31,541. Immucillin-H inhibits PNP with a Kd of 23 pM when only one of the three catalytic sites is occupied. Deuterium exchange occurs at 167 slow-exchange sites in 2 h when no catalytic site ligands are present. The substrate analogue and product prevented H/D exchange at 10 of the sites. Immucillin-H protected 32 protons from exchange at full saturation. When one of the three subunits of the homotrimer is filled with immucillin-H, and 27 protons are protected from exchange in all three subunits. Deuterium incorporation in peptides from residues 132-152 decreased in all complexes of PNP. The rate and/or extent of deuterium incorporation in peptides from residues 29-49, 50-70, 81-98, and 112-124 decreased only in the complex with the transition state analogue. The peptide-specific H/D exchange demonstrates that (1) the enzyme is most compact in the complex with immucillin-H, and (2) filling a single catalytic site of the trimer reduces H/D exchange in the same peptides in adjacent subunits. The peptides most highly influenced by the inhibitor surround the catalytic site, providing evidence for reduced protein dynamic motion caused by the transition state analogue.     

The structure of human 5'-deoxy-5'-methylthioadenosine phosphorylase at 1.7 A resolution provides insights into substrate binding and catalysis.

BACKGROUND: 5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAP) catalyzes the reversible phosphorolysis of 5'-deoxy-5'-methylthioadenosine (MTA) to adenine and 5-methylthio-D-ribose-1-phosphate. MTA is a by-product of polyamine biosynthesis, which is essential for cell growth and proliferation. This salvage reaction is the principle source of free adenine in human cells. Because of its importance in coupling the purine salvage pathway to polyamine biosynthesis MTAP is a potential chemotherapeutic target. RESULTS: We have determined the crystal structure of MTAP at 1.7 A resolution using multiwavelength anomalous diffraction phasing techniques. MTAP is a trimer comprised of three identical subunits. Each subunit consists of a single alpha/beta domain containing a central eight-stranded mixed beta sheet, a smaller five-stranded mixed beta sheet and six alpha helices. The native structure revealed the presence of an adenine molecule in the purine-binding site. The structure of MTAP with methylthioadenosine and sulfate ion soaked into the active site was also determined using diffraction data to 1.7 A resolution. CONCLUSIONS: The overall quaternary structure and subunit topology of MTAP are similar to mammalian purine nucleoside phosphorylase (PNP). The structures of the MTAP-ligand complexes provide a map of the active site and suggest possible roles for specific residues in substrate binding and catalysis. Residues accounting for the differences in substrate specificity between MTAP and PNP are also identified. Detailed information about the structure and chemical nature of the MTAP active site will aid in the rational design of inhibitors of this potential chemotherapeutic target. The MTAP structure represents the first structure of a mammalian PNP that is specific for 6-aminopurines.     

Methylthioinosine phosphorylase from Pseudomonas aeruginosa. Structure and annotation of a novel enzyme in quorum sensing

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 ? 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.     

Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.     

A transition state in pieces: major contributions of entropic effects to ligand binding by adenosine deaminase.

Nebularine undergoes hydration at the active site of adenosine deaminase, in a reaction analogous to a partial reaction in the displacement of ammonia from adenosine by water, to generate an inhibitory complex that captures much of the binding affinity expected of an ideal transition-state analogue. Enzyme affinities of several compounds related to nebularine 1,6-hydrate, and to its stable analog 2'-deoxycoformycin, were compared in an effort to identify the structural origins of strong binding. Binding of the stable transition-state analog inhibitor 2'-deoxycoformycin was rendered 9.8 kcal/mol less favorable by removal of substituent ribose, 9.7 kcal/mol less favorable by inversion of the 8-hydroxyl substituent of the diazepine ring, and 10.0 kcal/mol less favorable by removal of atoms 4-6 of the diazepine ring. Binding of the unstable transition-state analog nebularine hydrate was rendered at least 9.9 kcal/mol less favorable by removal of the 6-hydroxyl group and 10.2 kcal/mol less favorable by removal of atoms 1-3 of the pyrimidine ring. In each case, the enzyme exhibited only modest affinity (Kd greater than or equal to 10(-2) M) for the "missing piece", indicating that incorporation of 2 binding determinants within a single molecule permits an additional 7-12 kcal/mol of intrinsic binding energy to be manifested as observed binding energy. These results are consistent with earlier indications that adenosine deaminase may use 10.5 kcal/mol of the intrinsic free energy of binding of the two substrates to place them in positions appropriate for reaction at the active site, overcoming the unfavorable entropy change of -35 eu for the equilibrium of 1,6-hydration of purine ribonucleoside and reducing the equilibrium constant for attainment of the transition state in deamination of adenosine. Thus, adenosine deaminase may achieve up to 8 orders of magnitude of its catalytic power by converting the nonenzymatic, bimolecular, hydration reaction to a monomolecular reaction at its active site. Several new 6-substituted 1,6-dihydropurine ribonucleosides, prepared by photoaddition of formate and by low-temperature addition of organolithium reagents to a derivative of purine ribonucleoside, exhibited Ki values of 9-1400 microM against adenosine deaminase, in accord with the active site's considerable tolerance of bulky leaving groups in substrates. Inhibition by one diastereomer of 6-carboxy-1,6-dihydropurine ribonucleoside was found to be time-dependent, progressing from a weakly bound to a more strongly bound complex.     

Thermodynamic analysis of binding between mouse major urinary protein-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole

The mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT) binds to an occluded, nonpolar cavity in the mouse major urinary protein-I (MUP-I). The thermodynamics of this interaction have been characterized using isothermal titration calorimetry (ITC). MUP-I-SBT binding is accompanied by a large favorable enthalpy change (DeltaH = -11.2 kcal/mol at 25 degrees C), an unfavorable entropy change (-TDeltaS = 2.8 kcal/mol at 25 degrees C), and a negative heat capacity change [DeltaC(p)() = -165 cal/(mol K)]. Thermodynamic analysis of binding between MUP-I and several 2-alkyl-4,5-dihydrothiazole ligands indicated that the alkyl chain contributes more favorably to the enthalpy and less favorably to the entropy of binding than would be expected on the basis of the hydrophobic desolvation of short-chain alcohols. However, solvent transfer experiments indicated that desolvation of SBT is accompanied by a net unfavorable change in enthalpy (DeltaH = +1.0 kcal/mol) and favorable change in entropy (-TDeltaS = -1.8 kcal/mol). These results are discussed in terms of the possible physical origins of the binding thermodynamics, including (1) hydrophobic desolvation of both the protein and the ligand, (2) formation of a buried water-mediated hydrogen bond network between the protein and ligand, (3) formation of strong van der Waals interactions, and (4) changes in the structure, dynamics, and/or hydration of the protein upon binding.     

Immucillins as antibiotics for T-cell proliferation and malaria

The genetic deficiency of human PNP causes a specific immunodeficiency by inducing apoptosis in dividing T-cells. Powerful inhibitors of PNP have been designed from the experimental determination of the transition state structure of PNPs. The Immucillins are transition state analogue inhibitors with Kd values as low as 7 pM. In the presence of deoxyguanosine the Immucillins kill activated human T-cells but not other cell types. The Immucillins are orally available and of low toxicity to mice. Immucillins also inhibit PNP from Plasmodium falciparum. Parasites cultured in human erythrocytes are killed by purine starvation in the presence of Immucillins and can be rescued by hypoxanthine.     

Inhibition and Structure of Toxoplasma gondii Purine Nucleoside Phosphorylase

The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside phosphorylase (TgPNP) stability and crystallized TgPNP with phosphate and immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of 370 pM, the highest affinity of 11 immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared to Plasmodium falciparum PNP, large substituents surrounding the 5′-hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discrimination against large 5′ groups is consistent with the inability of TgPNP to catalyze the phosphorolysis of 5′-methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2′-hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP. This first crystal structure of TgPNP describes the basis for discrimination against 5′-methylthioinosine and similarly 5′-hydroxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa.