Promoting vibrations in human purine nucleoside phosphorylase. A molecular dynamics and hybrid quantum mechanical/molecular mechanical study

Crystallographic studies of human purine nucleoside phosphorylase (hPNP) with several transition-state (TS) analogues in the immucillin family showed an unusual geometric arrangement of the atoms O-5', O-4', and O(P), the nucleophilic phosphate oxygen, lying in a close three-oxygen stack. These observations were corroborated by extensive experimental kinetic isotope effect analysis. We propose that protein-facilitated dynamic modes in hPNP cause this stack, centered on the ribosyl O-4' oxygen, to squeeze together and push electrons toward the purine ring, stabilizing the oxacarbenium character of the TS. As the N-ribosidic bond is cleaved during the reaction, the pK(a) values of N-7 and O-6 increase by the electron density expelled by the oxygen-stack compression toward the purine ring. Increased electron density in the purine ring improves electrostatic interactions with nearby residues and facilitates the abstraction of a proton from a solvent proton or an unidentified general acid, making the purine a better leaving group, and accelerating catalysis. Classical and mixed quantum/classical molecular dynamics (MD) simulations of the Michaelis complex of hPNP with the substrates guanosine and phosphate were performed to assess the existence of protein-promoting vibrations (PPVs). Analogous simulations were performed for the substrates in aqueous solution. In the catalytic site, the O-5', O-4', and O(P) oxygens vibrate at frequencies of ca. 125 and 465 cm(-1), as opposed to 285 cm(-1) in the absence of hPNP. The hybrid quantum mechanical/molecular mechanical method was used to assess whether this enzymatic vibration pushing the oxygens together is coupled to the reaction coordinate, and thus has a direct positive impact on catalysis. The potential energy surface for the phosphorolysis reaction for several snapshots taken from the classical MD simulation showed substantial differences in oxygen compression. Our calculations showed the existence of PPVs coupled to the reaction coordinate, which effect electronic alterations in the active site by pushing the three oxygen centers together in proximity, and accelerate substrate turnover in the phosphorolysis reaction catalyzed by hPNP.     

Binding thermodynamics and interaction patterns of human purine nucleoside phosphorylase-inhibitor complexes from extensive free energy calculations

Journal of Computer-Aided Molecular Design - Human purine nucleoside phosphorylase (hPNP) plays a significant role in the catabolism of deoxyguanosine. The trimeric protein is an important target...     

Single Tryptophan of Disordered Loop from Plasmodium falciparum Purine Nucleoside Phosphorylase: Involvement in Catalysis and Microenvironment

Among various tropical diseases, malaria is a major life-threatening disease caused by Plasmodium parasite. Plasmodium falciparum is responsible for the deadliest form of malaria, so-called cerebral malaria. Purine nucleoside phosphorylase from P. falciparum is a homohexamer containing single tryptophan residue per subunit that accepts inosine and guanosine but not adenosine for its activity. This enzyme has been exploited as drug target against malaria disease. It is important to draw together significant knowledge about inherent properties of this enzyme which will be helpful in better understanding of this drug target. The enzyme shows disorder to order transition during catalysis. The single tryptophan residue residing in conserved region of transition loop is present in purine nucleoside phosphorylases throughout the Plasmodium genus. This active site loop motif is conserved among nucleoside phosphorylases from apicomplexan parasites. Modification of tryptophan residue by N-bromosuccinamide resulted in complete loss of activity showing its importance in catalysis. Inosine was not able to protect enzyme against N-bromosuccinamide modification. Extrinsic fluorescence studies revealed that tryptophan might not be involved in substrate binding. The tryptophan residue localised in electronegative environment showed collisional and static quenching in the presence of quenchers of different polarities.     

Structure-activity relationships for a collection of structurally diverse inhibitors of purine nucleoside phosphorylase

Values of inhibition constants, Ki, and concentrations required for 50% inhibition, IC50, for a collection of structurally diverse competitive inhibitors of calf spleen purine nucleoside phosphorylase have been determined employing inosine as substrate. These values have been employed to create predictive quantitative structure-activity relationships (QSAR) which link structure to values of Ki and IC50. These QSAR models have substantial power to predict values and the associated uncertainties for Ki and IC50 for unknown, structurally diverse inhibitors of purine nucleoside phosphorylase.     

The existance of a group translocation transport mechanism in animal cells: uptake of the ribose moiety of inosine.

After exposure to inosine, transport-competent plasma membrane vesicles isolated from SV-40-transformed Bal/c 3T3 cells accumulate intravesicular ribose 1-PO4 at a concentration 200-fold greater than the extravesicular concentration. An analysis of the purine nucleoside phosphorylase activity distribution in various subcellular fractions, relative to other enzyme activities, indicated the presence of plasma membrane-associated purine nucleoside phosphorylase activity. The plasma membrane vesicles appear relatively impermeable to hypoxanthine. However, hypoxanthine, which is a competitive inhibitor of the transport reaction, is the only compound tested capable of mediating efflux of already accumulated ribose 1-PO4. In addition, hypoxanthine does not result in the efflux of transported uridine which is accumulated in these membrane vesicles as uridine. Exogenous ribose 1-PO4 neither results in counterflow nor does it inhibit the original uptake reaction. The following transport reaction is proposed: uptake occurs by group translocation, mediated by membrane-localized purine nucleoside phosphorylase. The data are consistent with sites for inosine and hypoxanthine being on the outer membrane surface whereas the ribose 1-PO4 site is only on the inner surface.     

Structural Insight into the Binding of Hypoxanthine with Purine Nucleoside Phosphorylase from Streptococcus mutants

Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway and an attractive target for drug design. The crystal structure of Streptococcus mutants purine nucleoside phosphorylase(Smu PNP) has been solved by molecular replacement at 1.80  resolution and refined to R factors of 19.9%/23.7%(Rcryst/Rfree) . Sequence alignment and structural comparison show that Smu PNP has more similarity with PNPs isolated from human and malarial sources than the bacterial PNPs. The structure complexed with hypoxanthine(HPA) and sulfate ion was solved at 2.24  resolution and refined to R factors of 21.6%/24.1%(Rcryst/Rfree) . It is interesting to note that the resulting electron density indicated the product,HPA,presents in the active site although inosine was included in the crystallization mixture with Smu PNP. Asn233 and Glu191 are the important residues for ligand binding and recognition. Comparison with PNPs from different species gives detailed information about binding of small molecules on the active site,which is important for the studies of enzymatic mechanism and rational design of specific inhibitors for PNPs.     

Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base‐Modified Nucleosides

A wide range of natural purine analogues was used as probe to assess the mechanism of recognition by the wild‐type (WT) E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant. The results were analyzed from viewpoint of the role of the Ser90 residue and the structural features of the bases. It was found that the Ser90 residue of the PNP 1) plays an important role in the binding and activation of 8‐aza‐7‐deazapurines in the synthesis of their nucleosides, 2) participates in the binding of α‐D ‐pentofuranose‐1‐phosphates at the catalytic site of the PNP, and 3) catalyzes the dephosphorylation of intermediary formed 2‐deoxy‐α‐D ‐ribofuranose‐1‐phosphate in the trans‐2‐deoxyribosylation reaction. 5‐Aza‐7‐deazaguanine manifested excellent substrate activity for both enzymes, 8‐amino‐7‐thiaguanine and 2‐aminobenzothiazole showed no substrate activity for both enzymes. On the contrary, the 2‐amino derivatives of benzimidazole and benzoxazole are substrates and are converted into the N1‐ and unusual N2‐glycosides, respectively. 9‐Deaza‐5‐iodoxanthine showed moderate inhibitory activity of the WT E. coli PNP, whereas 9‐deazaxanthine and its 2′‐deoxyriboside are weak inhibitors.     

Functional concerted motions in the bovine serum retinol-binding protein

The large concerted motions in the apo/holo bovine serum retinol- binding protein were studied using molecular dynamics simulation and 'essential dynamics' analysis. Initially, concerted motions were calculated from conformational differences between various crystal structures. The dynamic behaviour of the protein in the configurational space directions, described by these concerted motions, is analysed. This reveals that the large backbone dynamics of the protein is not influenced by the presence of retinol. Study of free retinol dynamics and retinol in the retinol binding site reveals that the protein binds retinol in a favourable conformation, as opposed to what has been previously described for the bovine cellular retinol-binding protein.     

Design and divergent synthesis of aza nucleosides from a chiral imino sugar

Several novel nucleoside analogues as potential inhibitors of glycosidases and purine nucleoside phosphorylase (PNP) have been synthesized via selective coupling of an appropriate nucleobase at different positions of an orthogonally protected imino sugar as a common precursor. This synthetic strategy offers a straightforward protocol for the assembly of imino sugar containing nucleosides, establishing a new repertoire of molecules as potential therapeutics.     

Synthesis of a transition state analogue inhibitor of purine nucleoside phosphorylase via the Mannich reaction

[reaction: see text] The expeditious convergent synthesis of the potent human purine nucleoside phosphorylase inhibitor DADMe-Immucillin-G (3) was achieved via the Mannich reaction. The Mannich chemistry of a series of deazapurines and amine hydrochlorides was also investigated.     

Binding causes the remote [5'-3H]thymidine kinetic isotope effect in human thymidine phosphorylase

The remote 5'-3H V/K kinetic isotope effect (KIE) observed in human thymidine phosphorylase (6.1%) is significantly larger than can be explained by the reaction chemistry. One hypothesis connects the 5'-3H KIE in purine nucleoside phosphorylase to that enzyme's SN1 transition state. The transition state of thymidine phosphorylase, however, is an SN2 nucleophilic displacement. Here we report equilibrium binding isotope effects sufficiently large to explain the presence of this substantial KIE in thymidine phosphorylase.     

Determination of purine nucleosides and their bases by high-performance liquid chromatography using co-immobilized enzyme reactors

A selective and sensitive assay of inosine, guanosine, hypoxanthine, guanine and xanthine by high-performance liquid chromatography with immobilized enzyme reactors was developed. The separation was achieved on a Capcell Pak C18 column (15 cm x 0.46 cm I.D.) with a mobile phase of 0.1 M phosphate buffer (pH 8.0) containing 7 mM sodium 1-hexanesulphonate and 0.1 mM p-hydroxyphenylacetic acid. The fluorimetric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of these compounds, which were oxidized to yield hydrogen peroxide in the presence of immobilized enzyme (purine nucleoside phosphorylase, guanase and xanthine oxidase). Enzyme reactions occurred sufficiently without post-column addition of reagents. Enzymes that catalysed the conversion of purine compounds were co-immobilized on aminopropyl controlled-pore glass packed in stainless-steel tubing. The detection limits were 30-200 pg per injection.     

Simplex Optimization for the Simultaneous HPLC Assay of the Activities of Purine-Nucleoside Phosphorylase and Hypoxanthine-Guanine Phosphoribosyl Transferase

Abstract

The modified sequential simplex procedure is shown to be effective for maximizing a complex enzyme assay. The optlmum levels for two factors, pH and substrate concentration, for a coupled enzyme assay of hypoxanthine-quanlne phosphor Ibosy I-transferase (HGPRTase) and purine nucleoside phosphorylase (PNPase) were found by searching a factor space made up of these variables. The performance Index or response to be optimized was a function of the product of the two single activities. The maximum activity for this function was found at a pH of 7.9 and a concentration of Inosine in the reaction mix of 0.84 mM.     

Pyrimidine nucleoside phosphorylase assay by automated high-performance liquid chromatography

A new assay for pyrimidine nucleoside phosphorylase is reported. This method utilizes an isocratic reversed-phase high-performance liquid chromatographic system for separation of nucleosides and bases. Product detection is accompanied by ultraviolet monitoring and radioactive flow detection. Use of an automated sample injector allows for the analysis of a series of samples, with data recorded onto a microprocessor-based cassette recorder. Data can then be downloaded into computer memory. The velocity of uridine phosphorylase (E.C. 2.4.2.3) was a linear function of enzyme concentration. The Michaelis constant for uridine at pH 8.0 was found to be in close agreement with the value obtained by a thin-layer chromatographic assay method.     

L-Enantiomers of transition state analogue inhibitors bound to human purine nucleoside phosphorylase

Human purine nucleoside phosphorylase (PNP) was crystallized with transition-state analogue inhibitors Immucillin-H and DADMe-Immucillin-H synthesized with ribosyl mimics of l-stereochemistry. 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.     

Acyclic immucillin phosphonates: second-generation inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase

Plasmodium falciparum, the primary cause of deaths from malaria, is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. Here, we present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.     

Frontispiece: Analysis of the Dynamics of the Human Growth Hormone Secretagogue Receptor Reveals Insights into the Energy Landscape of the Molecule

G protein-coupled receptors initiate signal transduction in response to ligand binding. Growth hormone secretagogue receptor (GHSR), the focus of this study, binds the 28 residue peptide ghrelin. While structures of GHSR in different states of activation are available, dynamics within each state have not been investigated in depth. We analyze long molecular dynamics simulation trajectories using “detectors” to compare dynamics of the apo and ghrelin-bound states yielding timescale-specific amplitudes of motion. We identify differences in dynamics between apo and ghrelin-bound GHSR in the extracellular loop 2 and transmembrane helices 5–7. NMR of the GHSR histidine residues reveals chemical shift differences in these regions. We evaluate timescale specific correlation of motions between residues of ghrelin and GHSR, where binding yields a high degree of correlation for the first 8 ghrelin residues, but less correlation for the helical end. Finally, we investigate the traverse of GHSR over a rugged energy landscape via principal component analysis.     

Catalytic mechanism of glycosyltransferases: hybrid quantum mechanical/molecular mechanical study of the inverting N-acetylglucosaminyltransferase I

The Golgi glycosyltransferase, N-acetylglucosaminyltransferase I (GnT-I), catalyzes the transfer of a GlcNAc residue from the donor UDP-GlcNAc to the C2-hydroxyl group of a mannose residue in the trimannosyl core of the Man5GlcNAc2-Asn-X oligosaccharide. The catalytic mechanism of GnT-I was investigated using a hybrid quantum mechanical/molecular mechanical (QM/MM) method with a QM part containing 88 atoms treated with density functional theory (DFT) at the BP/TZP level. The remaining parts of a GnT-I complex, altogether 5633 atoms, were modeled using the AMBER molecular force field. A theoretical model of a Michaelis complex was built using the X-ray structure of GnT-I in complex with the donor having geometrical features consistent with kinetic studies. The QM(DFT)/MM model identified a concerted SN2-type of transition state with D291 as the catalytic base for the reaction in the enzyme active site. The TS model features nearly simultaneous nucleophilic addition and dissociation steps accompanied by the transfer of the nucleophile proton Hb2 to the catalytic base D291. The structure of the TS model is characterized by the Ob2-C1 and C1-O1 bond distances of 1.912 and 2.542 A, respectively. The activation energy for the proposed reaction mechanism was estimated to be approximately 19 kcal mol-1. The calculated alpha-deuterium kinetic isotope effect of 1.060 is consistent with the proposed reaction mechanism. Theoretical results also identified interactions between the Hb6 and beta-phosphate oxygen of the UDP and a low-barrier hydrogen bond between the nucleophile and the catalytic base D291. It is proposed that these interactions contribute to a stabilization of TS. This modeling study provided detailed insight into the mechanism of the GlcNAc transfer catalyzed by GnT-I, which is the first step in the conversion of high mannose oligosaccharides to complex and hybrid N-glycan structures.     

Determination of adenosine deaminase and purine nucleoside phosphorylase activities using high-performance liquid chromatographic and radiochromatographic methods

Two methods for the determination of adenosine deaminase and purine nucleoside phosphorylase activities were compared. The high-performance liquid chromatographic (HPLC) technique used separation on a reversed-phase silica column and exhibited adequate sensitivity and a markedly higher rate of analysis compared with that of the paper radiochromatographic method. Correlation analysis of the results obtained by the two methods on a set of lymphoid cells from 25 patients with lympho-proliferative disorders confirmed the utility of the HPLC technique in clinical investigations.     

Phosphate activation in the ground state of purine nucleoside phosphorylase

Phosphate and ribose 1-phosphate (R1P) bound to human purine nucleoside phosphorylase (PNP) have been studied by FTIR spectroscopy for comparison with phosphate bound with a transition state analogue. Bound phosphate is dianionic but exists in two distinct binding modes with similar binding affinities. The phosphate of bound R1P is also dianionic. Bound R1P slowly hydrolyzes to ribose and phosphate even in the absence of nucleobase. The C-OP bond is cleaved in bound R1P, the same as in the PNP-catalyzed reaction. Free R1P undergoes both C-OP and CO-P solvolysis. A hydrogen bond to one P-O group is stronger than those to the other two P-O groups in both the PNP.R1P complex and in one form of the PNP.PO4 complex. The average hydrogen bond strength to the PO bonds in the PNP.R1P complex is less than that in water but stronger than that in the PNP.PO4 complex. Hydrolysis of bound R1P may be initiated by distortion of the phosphate moiety in bound R1P. The unfavorable interactions on the phosphate moiety of bound R1P are relieved by dissociation of R1P from PNP or by hydrolysis to ribose and phosphate. The two forms of bound phosphate in the PNP.PO4 complex are interpreted to be phosphate positioned as the product in the nucleoside synthesis direction and as the reactant in the phosphorolysis reaction; their interconversion can occur by the transfer of a proton from one PO bond to another. The electronic structure of phosphate bound with a transition state analogue differs substantially from that in the Michaelis complexes.