Site-Directed Mutagenesis in Basic Amino Acid Residues of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed. No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V _max/K _mADP. These results indicate that Arg76 is not involved in CO₂ binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action [Herrera et al. (1993) J. Protein Chem. 12, 413–418]. These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate [Bazaes et al. (1995), FEBS Lett. 360, 207–210], does not perform an essential function for the enzyme activity.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

N(4)-Tolyl-2-acetylpyridine thiosemicarbazones and their platinum(II,IV) and gold(III) complexes: cytotoxicity against human glioma cells and studies on the mode of action

Complexes [Au(2Ac4oT)Cl][AuCl₂] (1), [Au(H_py2Ac4mT)Cl₂]Cl·H₂O (2), [Au(H_py2Ac4pT)Cl₂]Cl (3), [Pt(H2Ac4oT)Cl]Cl (4), [Pt(2Ac4mT)Cl]·H₂O (5), [Pt(2Ac4pT)Cl] (6) and [Pt(L)Cl₂OH], L = 2Ac4mT (7), 2Ac4oT (8), 2Ac4pT (9) were prepared with N(4)-ortho- (H2Ac4oT), N(4)-meta- (H2Ac4mT) and N(4)-para- (H2Ac4pT) tolyl-2-acetylpyridine thiosemicarbazone. The cytotoxic activities of all compounds were assayed against U-87 and T-98 human malignant glioma cell lines. Upon coordination cytotoxicity improved in 2, 5 and 8. In general, the gold(III) complexes were more cytotoxic than those with platinum(II,IV). Several of these compounds proved to be more active than cisplatin and auranofin used as controls. The gold(III) complexes probably act by inhibiting the activity of thioredoxin reductase enzyme whereas the mode of action of the platinum(II,IV) complexes involves binding to DNA. Cells treated with the studied compounds presented morphological changes such as cell shrinkage and blebs formation, which indicate cell death by apoptosis induction.     

Computational comparison of the kinetic stabilities of diamino- and diamidocarbenes in the 1,2-H shift reaction

In this study, we performed several DFT, MP2, and BD(T) calculations on the 1,2-H shift reactions of two diaminocarbenes (1, 2) and a diamidocarbene (3) using the Gaussian 09 program. In Gaussian 09, the BD(T) method keyword requests a Brueckner doubles calculation including a perturbative triples contribution. Although N-heterocyclic carbenes (NHC) are typically known for their exceptional σ-donor abilities, recent studies have indicated that π-interactions also play a role in the bonding between NHCs and transition metals or BX₃ (X = H, OH, NH₂, CH₃, CN, NC, F, Cl, and Br) (Nemcsok et al. Organomet 23:3640–3646, 2004, Esrafili. J Mol Model 18:2003–2011, 2012). In order to study the importance of π-interactions between carbenes and transition metals, Hobbs and co-workers (Hobbs et al. New J Chem 34:1295–1308, 2010) focused on the synthesis of NHCs with reduced-energy lowest unoccupied molecular orbitals. By introducing an oxalamide moiety into the heterocyclic backbone, they found the resulting carbene possessed higher electrophilicity than usual NHCs. According to our results, the N,N'-diamidocarbene should be more stable than the diaminocarbenes with respect to the 1,2-H shift reaction.

Di- and polynuclear silver(I) saccharinate complexes of tertiary diphosphane ligands: synthesis, structures, in vitro DNA binding, and antibacterial and anticancer properties

A series of new silver(I) saccharinate (sac) complexes, [Ag₂(sac)₂(μ-dppm)H₂O]·H₂O (1), {[Ag₂(μ-sac)₂(μ-dppe)]·3H₂O·CH₂Cl₂} _n (2), [Ag₂(μ-sac)₂(μ-dppp)] _n (3), and [Ag(sac)(μ-dppb)] _n (4) [dppm is 1,1-bis(diphenylphosphino)methane, dppe is 1,2-bis(diphenylphosphino)ethane, dppp is 1,3-bis(diphenylphosphino)propane, and dppb is 1,4-bis(diphenylphosphino)butane], have been synthesized and characterized by C, H, N elemental analysis, IR spectroscopy, ¹H NMR, ¹³C NMR, and ³¹P NMR spectroscopy, electrospray ionization mass spectrometry, and thermogravimetry–differential thermal analysis. Single-crystal X-ray studies show that the diphosphanes act as bridging ligands to yield a dinuclear complex (1) and one-dimensional coordination polymers (2 and 4), whereas the sac ligand adopts a μ₂-N/O bridging mode in 2, and is N-coordinated in 1 and 4. The interaction of the silver(I) complexes with fish sperm DNA was investigated using UV–vis spectroscopy, fluorescence spectroscopy, and agarose gel electrophoresis. The binding studies indicate that the silver(I) complexes can interact with fish sperm DNA through intercalation, and complexes 1 and 3 have the highest binding affinity. The gel electrophoresis assay further confirms the binding of the complexes with the pBR322 plasmid DNA. The minimum inhibitory concentrations of the complexes indicate that complex 1 exhibits very high antibacterial activity against standard bacterial strains of Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, being much higher than those of AgNO₃, silver sulfadiazine, ciprofloxacin, and gentamicin. Moreover, complexes 1–3 exhibit very high cytotoxic activity against A549 and MCF-7 cancer cell lines, compared with AgNO₃ and cisplatin. The bacterial and cell growth inhibitions of the silver(I) complexes are closely related to their DNA binding affinities.     

Key role of hydrazine to the interaction between oxaloacetic against phosphoenolpyruvic carboxykinase (PEPCK): ONIOM calculations

The interactions between oxaloacetic (OAA) and phosphoenolpyruvic carboxykinase (PEPCK) binding pocket in the presence and absence of hydrazine were carried out using quantum chemical calculations, based on the two-layered ONIOM (ONIOM2) approach. The complexes were partially optimized by ONIOM2 (B3LYP/6-31G(d):PM6) method while the interaction energies between OAA and individual residues surrounding the pocket were performed at the MP2/6-31G(d,p) level of theory. The calculated interaction energies (INT) indicated that Arg87, Gly237, Ser286, and Arg405 are key residues for binding to OAA with the INT values of ?1.93, ?2.06, ?2.47, and ?3.16 kcal mol^?1, respectively. The interactions are mainly due to the formation of hydrogen bonding interactions with OAA. Moreover, using ONIOM2 (B3LYP/6-31G(d):PM6) applied on the PEPCKHS complex, two proton transfers were observed; first, the proton was transferred from the carboxylic group of OAA to hydrazine while the second one was from Asp311 to Lys244. Such reactions cause the generation of binding strength of OAA to the pocket via electrostatic interaction. The orientations of Lys243, Lys244, His264, Asp311, Phe333, and Arg405 were greatly deviated after hydrazine incorporation. These indicate that hydrazine plays an important role in terms of not only changing the conformation of the binding pocket, but is also tightly bound to OAA resulting in its conformation change in the pocket. The understanding of such interaction can be useful for the design of hydrazine-based inhibitor for antichachexia agents.

Driving and retarding forces in a chemical reaction

The reaction force F(ξ) is the negative gradient of the potential energy of a chemical process along the intrinsic reaction coordinate ξ. We extend the rigorous concept of F(ξ) to the “activation strain model” of Bickelhaupt et al., to formulate the “strain” force F _str(ξ) that retards a reaction and the “interaction” force F _int(ξ) that drives it. These are investigated for a group of Diels-Alder cycloadditions. The results fully support the interpretation of the minimum of F(ξ) as defining the beginning of the transition from deformed reactants to eventual products.     

Cysteine 265 Is in the Active Site of, But Is Not Essential for Catalysis by tRNA-Guanine Transglycosylase (TGT) from Escherichia coli

Site-directed mutagenesis and X-ray absorption spectroscopy studies have previously shown that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is a zinc metalloprotein and identified the enzymic ligands to the zinc [Chong et al. (1995), Biochemistry 34, 3694–3701; Garcia et al. (1966), Biochemistry 35, 3133–3139]. During these studies one mutant, TGT (C265A), was found to exhibit a significantly lower specific activity, but was not found to be involved in the zinc site. The present report demonstrates that TGT is inactivated by treatment with thiol reagents (e.g., DTNB, MMTS, and N-ethylmaleimide). Further, this inactivation is shown to be due to modification of cysteine 265. The kinetic parameters for the mutants TGT (C265A) and TGT (C265S), however, suggest that this residue is not performing a critical role in the TGT reaction. We conclude that cysteine 265 is in the active site of TGT, but is not performing a critical catalytic function. This conclusion is supported by the recent determination of the X-ray crystal structure of the TGT from Zymomonas mobilis [Romier et al. (1966), EMBO J. 15, 2850–2857], which reveals that the residue corresponding to cysteine 265 is distant from the putative catalytic site, but is in the middle of a region of the enzyme surface proposed to bind tRNA.     

Oxidation and reduction of sulfite contribute to susceptibility and detoxification of SO2 in Populus × canescens leaves

Key Message

The critical level for SO ₂ susceptibility of Populus × canescens is approximately 1.2 μL L ^?1 SO ₂ . Both sulfite oxidation and sulfite reduction and assimilation contribute to SO ₂ detoxification.

Abstract

In the present study, uptake, susceptibility and metabolism of SO₂ were analyzed in the deciduous tree species poplar (Populus × canescens). A particular focus was on the significance of sulfite oxidase (SO) for sulfite detoxification, as SO has been characterized as a safety valve for SO₂ detoxification in herbaceous plants. For this purpose, poplar plants were exposed to different levels of SO₂ (0.65, 0.8, 1.0, 1.2 μL L^?1) and were characterized by visible injuries and at the physiological level. Gas exchange parameters (stomatal conductance for water vapor, CO₂ assimilation, SO₂ uptake) of the shoots were compared with metabolite levels (sulfate, thiols) and enzyme activities [SO, adenosine 5′-phosphosulfate reductase (APR)] in expanding leaves (80–90 % expanded). The critical dosage of SO₂ that confers injury to the leaves was 1.2 μL L^?1 SO₂. The observed increase in sulfur containing compounds (sulfate and thiols) in the expanding leaves strongly correlated with total SO₂ uptake of the plant shoot, whereas SO₂ uptake rate was strongly correlated with stomatal conductance for water vapor. Furthermore, exposure to high concentration of SO₂ revealed channeling of sulfite through assimilatory sulfate reduction that contributes in addition to SO-mediated sulfite oxidation to sulfite detoxification in expanding leaves of this woody plant species.     

Dominant species, functional assemblages and frequency of equilibrium phases in late summer phytoplankton assemblages in Hungarian small shallow lakes

Late summer phytoplankton associations were studied qualitatively and quantitatively in 80 Hungarian lakes altogether (mostly shallow salt lakes, reservoirs, oxbows, gravel pit lakes). Equilibrium phases sensu Sommer et al. (1993) were found only in 17 lakes. Most of them were under some kind (high salt content or very low level of nutrients) of stress factor. It is concluded that environmental stress forces phytoplankton communities towards equilibrium. No relationship between occurrence of equilibria and trophic state was found. Species number of non-equilibrated lakes was almost three times as high as those in equilibrium. Of the 31 recently described (Reynolds et al., 2002) phytoplankton assemblages most of those were recognized that are likely to occur in shallow lakes. Separation of a functional group W _S from W2 for Synura dominated lakes is suggested. It seemed also necessary to raise a group (Y _Ph) for lakes dominated by Phacotus. Sorting of Dinophyta species into different already described functional groups is desirable.     

Combination of 1,4-naphthoquinone with benzothiazoles had selective algicidal effects against harmful algae

A series of naphthoquinone-benzothiazole conjugates were synthesized as algicides, and their efficacies against harmful algal blooming species, such as Chattonella marina, Heterosigma akashiwo and Cochlodinium polykrikoides, were examined. The introduction of substituted benzothiazole at the C2 position of 1,4-naphthoquinone (compounds 1–9) resulted in higher algicidal activity against C. polykrikoides than the C6 conjugates (compounds 10–20). On the other hand, of the C6 conjugates, compounds 11 and 12 exhibited better algicidal activity against H. akashiwo, C. marina, and C. polykrikoides than the C2 conjugates. Further structure-activity analysis indicated that a replacement of the methoxy groups with hydroxyl groups (compounds 21–26) decreased the algicidal activity significantly. Among the various synthetic naphthoquinonebezothiazole conjugates tested, compound 12 was found to affect the most significant decrease in the level of C. polykrikoides growth, with an IC₅₀ of 0.19 μM. Compound 11 was found to be the most potent inhibitor against H. akashiwo and C. polykrikoides, with IC₅₀ values of 0.32 and 0.12 μM, respectively. Overall, these results highlight a possible method for controlling and inhibiting red tide forming algae using NQ derivatives.     

A quantitative description of membrane current and its application to conduction and excitation in nerve

This article concludes a series of papers concerned with the flow of electric current through the surface membrane of a giant nerve fibre (Hodgkinet al., 1952,J. Physiol. 116, 424–448; Hodgkin and Huxley, 1952,J. Physiol. 116, 449–566). Its general object is to discuss the results of the preceding papers (Section 1), to put them into mathematical form (Section 2) and to show that they will account for conduction and excitation in quantitative terms (Sections 3–6).     

Genetic engineering of EcoRI mutants with altered amino acid residues in the DNA binding site: physicochemical investigations give evidence for an altered monomer/dimer equilibrium for the Gln144Lys145 and Gln144Lys145Lys200 mutants

We have genetically engineered the Arg200----Lys mutant, the Glu144Arg145----GlnLys double mutant, and the Glu144Arg145Arg200----GlnLysLys triple mutant of the EcoRI endonuclease in extension of previously published work on site-directed mutagenesis of the EcoRI endonuclease in which Glu144 had been exchanged for Gln and Arg145 for Lys [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. All these mutants carry modifications in the DNA binding site. Mutant EcoRI proteins were purified to homogeneity and characterized by physicochemical techniques. All mutants have a very similar secondary structure composition. However, whereas the Lys200 mutant is not impaired in its capacity to form a dimer, the Gln144Lys145 and Gln144Lys145Lys200 mutants have a very much decreased propensity to form a dimer or tetramer depending on concentration as shown by gel filtration and analytical ultracentrifugation. This finding may explain the results of isoelectric focusing experiments which show that these two mutants have a considerably more basic pI than expected for a protein in which an acidic amino acid was replaced by a neutral one. Furthermore, while wild-type EcoRI and the Lys200 mutant are denatured in an irreversible manner upon heating to 60 degrees C, the thermal denaturation process as shown by circular dichroism spectroscopy is fully reversible with the Gln144Lys145 double mutant and the Gln144Lys145Lys200 triple mutant. All EcoRI endonuclease mutants described here have a residual enzymatic activity with wild-type specificity, since Escherichia coli cells overexpressing the mutant proteins can only survive in the presence of EcoRI methylase. The detailed analysis of the enzymatic activity and specificity of the purified mutant proteins is the subject of the accompanying paper [Alves et al. (1989) Biochemistry (following paper in this issue)].     

Modeling the effect of H-bonding interactions and molecular packing on the molecular structure of [Ag(ethylnicotinate)2]NO3 complex

The gas phase molecular structure of a single isolated molecule of [Ag(Etnic)₂NO₃];1 where Etnic = Ethylnicotinate was calculated using B3LYP method. The H-bonding interaction between 1 with one (complex 2) and two (complex 3) water molecules together with the dimeric formula [Ag(Etnic)₂NO₃]₂;4 and the tetrameric formula [Ag(Etnic)₂NO₃]₄;5 were calculated using the same level of theory to model the effect of intermolecular interactions and molecular packing on the molecular structure of the titled complex. The H-bond dissociation energies of complexes 2 and 3 were calculated to be in the range of 12.220–14.253 and 30.106–31.055 kcal?mol^?1, respectively, indicating the formation of relatively strong H-bonds between 1 and water molecules. The calculations predict bidentate nitrate ligand in the case of 1 and 2, leading to distorted tetrahedral geometry around the silver ion with longer Ag–O distances in case of 2 compared to 1, while 3 has a unidentate nitrate ligand leading to a distorted trigonal planar geometry. The packing of two [Ag(Etnic)₂NO₃] complex units; 4 does not affect the molecular geometry around Ag(I) ion compared to 1. In the case of 5, the two asymmetric units of the formula [Ag(Etnic)₂NO₃] differ in the bonding mode of the nitrate group, where the geometry around the silver ion is distorted tetrahedral in one unit and trigonal planar in the other. The calculations predicted almost no change in the charge densities at the different atomic sites except at the sites involved in the C–H?O interactions as well as at the coordinated nitrogen of the pyridine ring.

Probing Allosteric Binding Sites of the Maize Endosperm ADP-Glucose Pyrophosphorylase

Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a nonnative, low-activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) reported previously by others revealed that several sulfate ions bind to each enzyme. These sites are also believed to interact with allosteric regulators such as inorganic phosphate and 3-phosphoglycerate (3-PGA). Several arginine (Arg) side chains contact the bound sulfate ions in the potato structure and likely play important roles in allosteric effector binding. Alanine-scanning mutagenesis was applied to the corresponding Arg residues in both the small and large subunits of maize endosperm AGPase to determine their roles in allosteric regulation and thermal stability. Steady-state kinetic and regulatory parameters were measured for each mutant. All of the Arg mutants examined—in both the small and large subunits—bound 3-PGA more weakly than the wild type (A₅₀ increased by 3.5- to 20-fold). By contrast, the binding of two other maize AGPase allosteric activators (fructose-6-phosphate and glucose-6-phosphate) did not always mimic the changes observed for 3-PGA. In fact, compared to 3-PGA, fructose-6-phosphate is a more efficient activator in two of the Arg mutants. Phosphate binding was also affected by Arg substitutions. The combined data support a model for the binding interactions associated with 3-PGA in which allosteric activators and inorganic phosphate compete directly.ADP-Glc pyrophosphorylase (AGPase), a key enzyme in starch biosynthesis, catalyzes the formation of ADP-Glc from ATP and Glc-1-P (G-1-P). Maize (Zea mays) AGPase, like nearly all higher plant homologs, is a highly regulated heterotetramer containing two small and two large subunits. By contrast, virtually all bacterial forms of the enzyme are homotetramers. Evidence from eight independent plant transgenic or genetic experiments (L.C. Hannah and T.W. Greene, unpublished data; Stark et al., 1992; Giroux et al., 1996; Smidansky et al., 2002, 2003; Sakulsingharoj et al., 2004; Obana et al., 2006; Wang et al., 2007) has shown that altering the allosteric properties and/or heat stability of AGPase can significantly increase starch content and starch turnover and, in turn, seed yield. Increased seed number giving rise to enhanced starch content occurs in some cases. Such observations have inspired efforts to understand AGPase regulation at a molecular level.Virtually all known AGPases are subject to allosteric activation and inhibition by various metabolites associated with the specific carbon utilization pathway of the organism. For example, the bacterial AGPase from Agrobacterium tumefaciens is activated by Fru-6-P (F-6-P) and inhibited by inorganic phosphate (P_i), whereas the Escherichia coli AGPase is activated by Fru-1,6-bisP but inhibited by AMP. Rhodospirillum rubrum AGPase is activated by both Fru-1,6-bisP and F-6-P, and inhibited by P_i, while Anabaena AGPase mimics plant AGPases in its activation by 3-phosphoglycerate (3-PGA) and inhibition by P_i. Using both chemical modification and site-directed mutagenesis, several Arg and Lys residues participating in allosteric regulation have been mapped to the C-terminal segments of the Anabaena and potato (Solanum tuberosum) tuber enzymes (Charng et al., 1994; Sheng et al., 1996; Ballicora et al., 1998, 2002).Unfortunately, only limited atomic-level structural data are available for AGPases. The three-dimensional structure of a bacterial homotetrameric enzyme from A. tumefaciens has recently been solved (Cupp-Vickery et al., 2008). Only one crystal structure is available for a higher plant AGPase: a nonnative, low-activity form of the enzyme from potato tuber (small subunit homotetramer; Jin et al., 2005). Although both structures reflect inactive conformations due to high concentrations of ammonium sulfate in the crystallization buffer, important information about potential substrate-binding sites was predicted by molecular modeling based on the known structures of thymidilyltransferases. While this class of enzymes likely binds sugar phosphates in the same manner as AGPases, thymidilyltransferases are not regulated allosterically. Both AGPase crystal structures suggest that the enzyme functions as a dimer of dimers, similar to the mechanism proposed for the Escherichia coli enzyme on the basis of ligand-binding studies (Haugen and Preiss, 1979). All available evidence leads to the conclusion that tetramers are required for AGPase catalytic activity.Both available AGPase crystal structures show two domains in each subunit: an N-terminal catalytic domain, which resembles previously reported pyrophosphorylase structures (Jin et al., 2005; Cupp-Vickery et al., 2008) and a C-terminal domain that makes strong hydrophobic interactions with the catalytic domain. In the potato small subunit homotetramer, two of the three bound sulfate ions (per monomer) are located in a crevice between the N- and C-terminal domains, separated by 7.24 Å. We have arbitrarily labeled these sites as sulfate 1 and sulfate 2, respectively. The third sulfate ion (in site 3) binds between two protein-adjacent monomers. When ATP is included in the crystallization buffer, two substrate molecules are bound in two of the four presumptive active sites, consistent with the notion that the protein functions as a dimer of dimers. On the other hand, one of the sulfate ions originally found in site 3 is lost when ATP is bound, despite the large distance between their respective binding sites. The A. tumefaciens AGPase homotetramer binds a single sulfate ion (per monomer) with 100% occupancy (Cupp-Vickery et al., 2008).All known allosteric regulators of higher plant AGPases contain one or more phosphate moieties. Because of their structural similarity, it is likely that the sulfate ions found in AGPase crystal structures bind in sites normally occupied by P_i or anionic, phosphorylated ligands such as F-6-P, G-6-P, and 3-PGA. Several studies suggest that all AGPase activators and inhibitors compete for binding to the same or closely adjacent sites within a subunit (Morell et al., 1988; Boehlein et al., 2008). Like P_i, sulfate reverses 3-PGA-mediated activation for the potato, A. tumefaciens, and maize enzymes (I_0.5 = 2.8 mm in the presence of 6 mm 3-PGA, potato tuber AGPase; I_0.5 = 20 mm in the presence of 2.5 mm 3-PGA, maize endosperm AGPase; Jin et al., 2005; S.K. Boehlein, unpublished data). In addition, both sulfate and P_i significantly affect maize AGPase thermal stability. For these reasons, we analyzed sulfate ion binding to the potato small subunit homotetramer to guide Ala-scanning mutagenesis studies on the analogous anion-binding sites within the heterotetrameric maize endosperm AGPase. Replacements were made in both the small and the large subunits of the maize endosperm AGPase. More conservative changes (Gln or Lys) were employed when Ala mutants displayed no catalytic activity. We chose not to create homology models of the maize subunits to help understand the behavior of Arg mutants. While computational models often predict core structures accurately, small details such as ligand-binding sites and subunit-subunit contacts are less reliable. This is particularly important for sulfate ion-binding site 3, which is located at the interface between two subunits. The problems are compounded by the lack of experimental data for an AGPase large subunit.Our studies revealed that altering any Arg residue that participates in a sulfate ion binding—either in the small or the large subunits of maize AGPase—drastically altered the enzyme''s overall allosteric properties. This indicates that effector-binding sites in both subunits function in concert in the native heterotetramer, reminiscent of their synergistic participation in catalysis. It also directly supports the notion that sulfate ion-binding sites are also involved in binding allosteric effectors. On the other hand, while mutations at all sulfate ion-binding sites affected allostery, substantial variation was observed for the different Arg side chains. Finally we note that while the various AGPases of plant and bacterial origin exhibit vastly different allosteric properties, presumably due to differing selection pressures over evolutionary time, single amino acid changes of the maize endosperm enzyme can create allosteric properties that mimic those exhibited by bacterial and other AGPases.     

The role of fluorine in stabilizing the bioactive conformation of dihydroorotate dehydrogenase inhibitors

Dihydroorotate dehydrogenase (DHODH) is an important drug target due to its prominent role in pyrimidine biosynthesis. Leflunomide and brequinar are two well-known DHODH inhibitors, which bind to the enzyme in the same pocket with different binding modes. We have recently realized a series of new inhibitors based on the 4-hydroxy-1,2,5-oxadiazole ring, whose activity profile was found to be closely dependent on the degree of fluorine substitution at the phenyl ring adjacent to the oxadiazole moiety; a positive influence of fluorine on the DHODH inhibitory potency was observed previously [Baumgartner et al. (2006) J Med Chem 49:1239–1247]. Potential energy surface scans showed that fluorine plays an important role in stabilizing the bioactive conformations; additionally, fluorine influences the balance between leflunomide-like and brequinar-like binding modes. These findings may serve as a guide to design more potent DHODH inhibitors.     

Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn

Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO₂. They are activated by Mn²⁺, a metal ion that coordinates to the protein through the ?-amino group of a lysine residue, the N_?-2-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the ?-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the ?-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn²⁺ in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn²⁺ affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn²⁺. In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.     

DNA repair genes polymorphism and lung cancer risk with the emphasis to sex differences

Polymorphisms in nucleotide and base excision repair genes are associated with the variability in the risk of developing lung cancer. In the present study, we investigated the polymorphisms of following selected DNA repair genes: XPC (Lys939Gln), XPD (Lys751Gln), hOGG1 (Ser326Cys) and XRCC1 (Arg399Gln), and the risks they present towards the development of lung cancer with the emphasis to gender differences within the Slovak population. We analyzed 761 individuals comprising 382 patients with diagnosed lung cancer and 379 healthy controls. Genotypes were determined by polymerase chain reaction/restriction fragment length polymorphism method. We found out statistically significant increased risk for lung cancer development between genders. Female carrying XPC Gln/Gln, XPC Lys/Gln+Gln/Gln and XRCC1 Arg/Gln, XRCC1 Arg/Gln+Gln/Gln genotypes had significantly increased risk of lung cancer corresponding to OR = 2.06; p = 0.04, OR = 1.66; p = 0.04 and OR = 1.62; p = 0.04, OR = 1.69; p = 0.02 respectively. In total, significantly increased risk of developing lung cancer was found in the following combinations of genotypes: XPD Lys/Gln+XPC Lys/Lys (OR = 1.62; p = 0.04), XRCC1 Gln/Gln+hOGG1 Ser/Ser (OR = 2.14; p = 0.02). After stratification for genders, the following combinations of genotype were found to be significant in male: XPD Lys/Gln+XPC Lys/Lys (OR = 1.87; p = 0.03), XRCC1 Arg/Gln+XPC Lys/Lys (OR = 4.52; p = 0.0007), XRCC1 Arg/Gln+XPC Lys/Gln (OR = 5.44; p < 0.0001). In female, different combinations of the following genotypes were found to be significant: XRCC1 Arg/Gln+hOGG1 Ser/Ser (OR = 1.98; p = 0.04), XRCC1 Gln/Gln+hOGG1 Ser/Ser (OR = 3.75; p = 0.02), XRCC1 Arg/Gln+XPC Lys/Gln (OR = 2.40; p = 0.04), XRCC1 Arg/Gln+XPC Gln/Gln (OR = 3.03; p = 0.04). We found out decreased cancer risk in genotype combinations between female patients and healthy controls: XPD Lys/Lys+XPC Lys/Gln (OR = 0.45; p = 0.02), XPD Lys/Gln+XPC Lys/Lys (OR = 0.32; p = 0.005), XPD Lys/Gln+XPC Lys/Gln (OR = 0.48; p = 0.02). Our results did not show any difference between pooled smokers and non-smokers in observed gene polymorphisms in the association to the lung cancer risk. However, gender stratification indicated the possible effect of heterozygous constitution of hOGG1 gene (Ser/Cys) on lung cancer risk in female non-smokers (OR = 0.20; p = 0.01) and heterozygous constitution of XPC gene (Lys/Gln) in male smokers (OR = 2.70; p = 0.01).     

Metal–drug synergy: new ruthenium(II) complexes of ketoconazole are highly active against Leishmania major and Trypanosoma cruzi and nontoxic to human or murine normal cells

In our ongoing search for new metal-based chemotherapeutic agents against leishmaniasis and Chagas disease, six new ruthenium–ketoconazole (KTZ) complexes have been synthesized and characterized, including two octahedral coordination complexes—cis,fac-[Ru^IICl₂(DMSO)₃(KTZ)] (1) and cis-[Ru^IICl₂(bipy)(DMSO)(KTZ)] (2) (where DMSO is dimethyl sulfoxide and bipy is 2,2′-bipyridine)—and four organometallic compounds—[Ru^II(η⁶-p-cymene)Cl₂(KTZ)] (3), [Ru^II(η⁶-p-cymene)(en)(KTZ)][BF₄]₂ (4), [Ru^II(η⁶-p-cymene)(bipy)(KTZ)][BF₄]₂ (5), and [Ru^II(η⁶-p-cymene)(acac)(KTZ)][BF₄] (6) (where en is ethylenediamine and acac is acetylacetonate); the crystal structure of 3 is described. The central hypothesis of our work is that combining a bioactive compound such as KTZ and a metal in a single molecule results in a synergy that can translate into improved activity and/or selectivity against parasites. In agreement with this hypothesis, complexation of KTZ with Ru^II in compounds 3–5 produces a marked enhancement of the activity toward promastigotes and intracellular amastigotes of Leishmania major, when compared with uncomplexed KTZ, or with similar ruthenium compounds not containing KTZ. Importantly, the selective toxicity of compounds 3–5 toward the leishmania parasites, in relation to human fibroblasts and osteoblasts or murine macrophages, is also superior to the selective toxicities of the individual constituents of the drug. When tested against Trypanosoma cruzi epimastigotes, some of the organometallic complexes displayed activity and selectivity comparable to those of free KTZ. A dual-target mechanism is suggested to account for the antiparasitic properties of these complexes.