Inhibition and Structure of Trichomonas vaginalis Purine Nucleoside Phosphorylase with Picomolar Transition State Analogues † (original) (raw)
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Biochemistry, 2002
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.
Biochemical and Biophysical Research Communications, 2010
Low molecular mass purine nucleoside phosphorylases (PNPs, E.C. 2.4.2.1) are homotrimeric enzymes that are tightly inhibited by immucillins. Due to the positive charge on the ribose like part (iminoribitol moiety) and protonation of the N7 atom of the purine ring, immucillins are believed to act as transition state analogues. Over a wide range of concentrations, immucillins bind with strong negative cooperativity to PNPs, so that only every third binding site of the enzyme is occupied (third-of-the-sites binding). 9-(5 0 ,5 0difluoro-5 0 -phosphonopentyl)-9-deazaguanine (DFPP-DG) shares with immucillins the protonation of the N7, but not the positive charge on the ribose like part of the molecule. We have previously shown that DFPP-DG interacts with PNPs with subnanomolar inhibition constant. Here, we report additional biochemical experiments to demonstrate that the inhibitor can be bound with the same K d ($190 pM) to all three substrate binding sites of the trimeric PNP, and a crystal structure of PNP in complex with DFPP-DG at 1.45 Å resolution, the highest resolution published for PNPs so far. The crystals contain the full PNP homotrimer in the asymmetric unit. DFPP-DG molecules are bound in superimposable manner and with full occupancies to all three PNP subunits. Thus the postulated third-of-the-sites binding of immucillins should be rather attribute to the second feature of the transition state, ribooxocarbenium ion character of the ligand or to the coexistence of both features characteristic for the transition state. The DFPP-DG/PNP complex structure confirms the earlier observations, that the loop from Pro57 to Gly66 covering the phosphatebinding site cannot be stabilized by phosphonate analogues. The loop from Glu250 to Gln266 covering the base-binding site is organized by the interactions of Asn243 with the Hoogsteen edge of the purine base of analogues bearing one feature of the postulated transition state (protonated N7 position).
Structural basis for inhibition of human PNP by immucillin-H
Biochemical and Biophysical Research Communications, 2003
Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and de-15 oxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation. This work reports on the 16 crystallographic study of the complex of human PNP-immucillin-H (HsPNP-ImmH) solved at 2.6 A A resolution using synchrotron 17 radiation. Immucillin-H (ImmH) inhibits the growth of malignant T-cell lines in the presence of deoxyguanosine without affecting 18 non-T-cell tumor lines. ImmH inhibits activated normal human T cells after antigenic stimulation in vitro. These biological effects of 19 ImmH suggest that this agent may have utility in the treatment of certain human diseases characterized by abnormal T-cell growth 20 or activation. This is the first structural report of human PNP complexed with immucillin-H. The comparison of the complex 21 HsPNP-ImmH with recent crystallographic structures of human PNP explains the high specificity of immucillin-H for human PNP. 22
Journal of the American Chemical Society, 2008
Human purine nucleoside phosphorylase (PNP) was crystallized with transition state analogue inhibitors Immucillin-H and DADMe-Immucillin-H synthesized with ribosyl mimics of Lstereochemistry. The inhibitors demonstrate that major driving forces for tight binding of these analogues are the leaving group interaction and the cationic mimicry of the transition state, even though large geometric changes occur with D-Immucillins and L-Immucillins bound to human PNP.
Biochemistry, 2003
Stable chemical analogues of enzymatic transition states are imperfect mimics since they lack the partial bond character of the transition state. We synthesized structural variants of the Immucillins as transition state analogues for purine nucleoside phosphorylase and characterized them with the enzyme from Mycobacterium tuberculosis (MtPNP). PNPs form transition states with ribooxacarbenium ion character and catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon between the enzymatically immobilized purine and phosphate nucleophiles. As bond-breaking progresses, carbocation character builds on the ribosyl group, the distance between the purine and the carbocation increases, and the distance between carbocation and phosphate anion decreases. Transition state analogues were produced with carbocation character and increased distance between the ribooxacarbenium ion and the purine mimics by incorporating a methylene bridge between these groups. Immucillin-H (ImmH), DADMe-ImmH, and DADMe-ImmG mimic the transition state of MtPNP and are slow-onset, tight-binding inhibitors of MtPNP with equilibrium dissociation constants of 650, 42, and 24 pM. Crystal structures of MtPNP complexes with ImmH and DADMe-ImmH reveal an ion-pair between the inhibitor cation and the nucleophilic phosphoryl anion. The stronger ion-pair (2.7 Å) is found with DADMe-ImmH. The position of bound ImmH resembles the substrate side of the transition state barrier, and DADMe-ImmH more closely resembles the product side of the barrier. The ability to probe both substrate and product sides of the transition state barrier provides expanded opportunities to explore transition state analogue design in N-ribosyltransferases. This approach has resulted in the highest affinity transition state analogues known for MtPNP.
Journal of Biological Chemistry, 2005
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 k cat for huPNP being 18-fold greater than k cat for PfPNP. This energetic binding difference between huPNP and PfPNP supports the k chem /k cat 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 Asp 206 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.
Acta Crystallographica Section F: Structural Biology Communications, 2018
Purine nucleoside phosphorylases (EC 2.4.2.1; PNPs) reversibly catalyze the phosphorolytic cleavage of glycosidic bonds in purine nucleosides to generate ribose 1-phosphate and a free purine base, and are key enzymes in the salvage pathway of purine biosynthesis. They also catalyze the transfer of pentosyl groups between purine bases (the transglycosylation reaction) and are widely used for the synthesis of biologically important analogues of natural nucleosides, including a number of anticancer and antiviral drugs. Potent inhibitors of PNPs are used in chemotherapeutic applications. The detailed study of the binding of purine bases and their derivatives in the active site of PNPs is of particular interest in order to understand the mechanism of enzyme action and for the development of new enzyme inhibitors. Here, it is shown that 7-deazahypoxanthine (7DHX) is a noncompetitive inhibitor of the phosphorolysis of inosine by recombinant Escherichia coli PNP (EcPNP) with an inhibition constant K i of 0.13 mM. A crystal of EcPNP in complex with 7DHX was obtained in microgravity by the counter-diffusion technique and the threedimensional structure of the EcPNP-7DHX complex was solved by molecular replacement at 2.51 Å resolution using an X-ray data set collected at the SPring-8 synchrotron-radiation facility, Japan. The crystals belonged to space group P6 1 22, with unit-cell parameters a = b = 120.370, c = 238.971 Å , and contained three subunits of the hexameric enzyme molecule in the asymmetric unit. The 7DHX molecule was located with full occupancy in the active site of each of the three crystallographically independent enzyme subunits. The position of 7DHX overlapped with the positions occupied by purine bases in similar PNP complexes. However, the orientation of the 7DHX molecule differs from those of other bases: it is rotated by $180 relative to other bases. The peculiarities of the arrangement of 7DHX in the EcPNP active site are discussed.
Proteins: Structure, Function, and Genetics, 1993
In the X-ray structure of the ternary staphylococcal nuclease-Ca2+-3',5'-pdTp complex, the conformation of the bound inhibitor 3',5'-pdTp is distorted by Lys-70* and Lys-71* from an adjacent molecule of the enzyme in the crystal lattice (Loll, P. Biochemistry 28:1539Biochemistry 28: -1548Biochemistry 28: , 1989. Since this interaction does not occur in solution, the NMR docking procedure has been used to correct this problem. Based on 8 Co2+-nucleus distances measured by paramagnetic effects on TI, and 9 measured and 45 lower limit interproton distances determined by 1D and 2D NOE studies of the ternary Ca2+ complex, the conformation of enzyme-bound 3',5'-pdTp is high-anti (x = 58 f 10") with a C2' endo/Ol' endo sugar pucker (6 = 143 f 29, (-1 synclinal about the C3'43' bond (e = 273 f 4"), trans, gauche about the C4'45' bond ( y = 301 +-29") and either (-1 or ( + 1 clinal about the C5'-05' bond (p = 92 2 8" or 274 2 3"). The structure of 3',5'-pdTp in the crystalline complex differs due to rotations about the C4'45' bond (y = 186 f 12", gauche, trans) and the C5'45' bond [p = 136 f lo", (+)
Proceedings of the National Academy of Sciences, 2013
Transition state analogs mimic the geometry and electronics of the transition state of enzymatic reactions. These molecules bind to the active site of the enzyme much tighter than substrate and are powerful noncovalent inhibitors. Immucillin-H (ImmH) and 4′deaza-1′-aza-2′-deoxy-9-methylene Immucillin-H (DADMe-ImmH) are picomolar inhibitors of human purine nucleoside phosphorylase (hPNP). Although both molecules are electronically similar to the oxocarbenium-like dissociative hPNP transition state, DADMe-ImmH is more potent than ImmH. DADMe-ImmH captures more of the transition state binding energy by virtue of being a closer geometric match to the hPNP transition state than ImmH. A consequence of these similarities is that the active site of hPNP exerts greater distortional forces on ImmH than on DADMe-ImmH to "achieve" the hPNP transition state geometry. By using magic angle spinning solid-state NMR to investigate stable isotope-labeled ImmH and DADMe-ImmH, we have explored the difference in distortional binding of these two inhibitors to hPNP. High-precision determinations of internuclear distances from NMR recoupling techniques, rotational echo double resonance, and rotational resonance, have provided unprecedented atomistic insight into the geometric changes that occur upon binding of transition state analogs. We conclude that hPNP stabilizes conformations of these chemically distinct analogs having distances between the cation and leaving groups resembling those of the known transition state.
Scientific Reports
Purine nucleoside phosphorylase (PNP) catalyses the cleavage of the glycosidic bond of purine nucleosides using phosphate instead of water as a second substrate. PNP from Escherichia coli is a homohexamer, build as a trimer of dimers, and each subunit can be in two conformations, open or closed. This conformational change is induced by the presence of phosphate substrate, and very likely a required step for the catalysis. Closing one active site strongly affects the others, by a yet unclear mechanism and order of events. Kinetic and ligand binding studies show strong negative cooperativity between subunits. Here, for the first time, we managed to monitor the sequence of nucleoside binding to individual subunits in the crystal structures of the wild-type enzyme, showing that first the closed sites, not the open ones, are occupied by the nucleoside. However, two mutations within the active site, Asp204Ala/Arg217Ala, are enough not only to significantly reduce the effectiveness of the enzyme, but also reverse the sequence of the nucleoside binding. In the mutant the open sites, neighbours in a dimer of those in the closed conformation, are occupied as first. This demonstrates how important for the effective catalysis of Escherichia coli PNP is proper subunit cooperation. Purine nucleoside phosphorylase (PNP, purine nucleoside orthophosphate ribosyl transferase, EC 2.4.2.1) has a crucial role in the purine salvage pathway 1. It catalyses the reversible phosphorolytic cleavage of the glycosidic bond of purine (2′-deoxy) nucleosides, generating the corresponding free base and (2′-deoxy) ribose 1-phosphate 1,2. The biologically active form of this enzyme, with only one known exception-PNP from Thermus thermophilus HB27 3 , is always oligomeric: homotrimers are characteristic mostly for mammals, while homohexamers are typical for most bacteria. The arrangement of subunits in the structure of a PNP hexamer is such that two of them donate two amino acids (His4 and Arg43 in E. coli) to each other, thus completing the active site of its neighbour, effectively forming a dimer which possesses an approximate 2-fold symmetry. Three such dimers are then arranged by a 3-fold symmetry axis to form a hexamer. The molecular mechanism by which this enzyme accomplishes its biological function is particularly intricate. In addition to the phosphorolysis being a two-substrate and two-product reversible reaction, with communication between monomers forming a dimer 1 , the mechanism is further complicated by the higher order communication, i.e. an allosteric cross-talk between dimers forming the hexamer 4,5. The way in which this allosteric communication is achieved is still unclear, but the complexity is reflected in an ever increasing number of different arrangements of active site conformations between monomers in the available crystal structures of hexameric PNPs 6. The catalytic mechanism of PNP, especially that of E. coli PNP, has been in the focus of our study for many years 2,5,7-16. It has been firmly established 5,14 that the initial step in the catalysis involves breaking a single α-helix