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J Feeney

Publications and source records attributed to J Feeney.

At least 91 records · Page 5Linked to original sources
PubMed1989

Interactions between inhibitors of dihydrofolate reductase.

The binding of substrates and inhibitors to dihydrofolate reductase was studied by steady-state kinetics and high-field 1H-n.m.r. spectroscopy. A series of 5-substituted 2,4-diaminopyrimidines were examined and were found to be 'tightly binding' inhibitors of the enzyme (Ki less than 10(-9) M). Studies on the binding of 4-substituted benzenesulphonamides and benzenesulphonic acids also established the existence of a 'sulphonamide-binding site' on the enzyme. Subsequent n.m.r. experiments showed that there are two binding sites for the sulphonamides on the enzyme, one of which overlaps the coenzyme (NADPH) adenine-ring-binding site. An examination of the pH-dependence of the binding of sulphonamides to the enzyme indicated the influence of an ionizable group on the enzyme that was not directly involved in the sulphonamide binding. The change in pKa value from 6.7 to 7.2 observed on sulphonamide binding suggests the involvement of a histidine residue, which could be histidine-28.

Benzenesulfonates↗
PubMed1989

NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases.

Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21----Leu and Asp 26----Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1H, 13C, and 31P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1H NMR studies of the Trp 21----Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 A away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21----Leu mutant indicate that the delicately poised equilibria can be perturbed. For example, in the case of the ternary complex formed between enzyme, trimethoprim, and NADP+, two almost equally populated conformations (forms I and II) are seen with the wild-type enzyme but only form II (the one in which the nicotinamide ring of the coenzyme is extended away from the enzyme structure and into the solvent) is observed for the mutant enzyme complex. It appears that the Trp 21----Leu substitution has a major effect on the binding of the nicotinamide ring of the coenzyme. For the Asp 26----Glu enzyme there is a change in the bound conformation of the substrate folate. Further indications that some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim come from the observation of a change in the dynamics of the bound trimethoprim molecule as seen from the increased rate of the flipping of the 13C-labeled benzyl ring and the increased rate of the N1-H bond breaking.

Cloning, Molecular↗
PubMed1989

Enzymic synthesis, chemical characterisation and Sda activity of GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4GlcNAc and GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc.

The tetrasaccharides GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc and GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3'-sialyllactose (NeuAc alpha 2-3 Gal beta 1-4Glc) and 3'-sialyl-N-acetyllactosamine (NeuAc alpha 2-3Gal beta 1-4GlcNAc). The structures of the products were established by methylation and 1H-500 MHz NMR spectroscopy. In Sda serological tests the product formed with 3'-sialyl-N-acetyllactosamine was highly active whereas that formed with 3'-sialyllactose had only weak activity.

Animals↗
PubMed1989

Dihydrofolate reductase: control of the mode of substrate binding by aspartate 26.

The complex of Lactobacillus casei dihydrofolate reductase with the substrate folate and the coenzyme NADP+ has been shown to exist in solution as a mixture of three slowly interconverting conformations whose proportions are pH-dependent and which differ in the orientation of the pteridine ring of the substrate in the binding site. The Asp26----Asn mutant of L. casei dihydrofolate reductase has been prepared by oligonucleotide-directed mutagenesis and studied by one- and two-dimensional 1H-NMR spectroscopy. NMR studies of the mutant enzyme--folate--NADP+ complex show that this exists to greater than 90% in a single conformation over the pH* range 5-7.1. The single conformation observed corresponds to conformation I (the 'methotrexate-like' conformation) of the wild-type enzyme--folate--NADP+ complex. These observations demonstrate that Asp26 is the ionizable group controlling the pH-dependence of the conformational equilibrium seen in the wild-type enzyme.

Amino Acid Sequence↗
PubMed1988

Identification of a novel oligosaccharide backbone structure with a galactose residue monosubstituted at C-6 in human foetal gastrointestinal mucins.

An oligosaccharide purified from a major penta- to hexa-saccharide fraction of human meconium glycoproteins has been shown by m.s. and n.m.r. analysis to have a novel backbone structure containing an internal galactose residue monosubstituted at C-6 by N-acetylglucosamine: (Formula: see text). This oligosaccharide may represent a biosynthetic product of a previously unrecognized N-acetylglucosaminyltransferase catalysing formation of a linear GlcNAc beta 1-6Gal sequence.

Carbohydrate Conformation↗
PubMed1988

Direct 19F NMR observation of the conformational selection of optically active rotamers of the antifolate compound fluoronitropyrimethamine bound to the enzyme dihydrofolate reductase.

The molecular basis of the binding of the lipophilic antifolate compound fluoronitropyrimethamine [2,4-diamino-5-(4-fluoro-3-nitrophenyl)-6-ethylpyrimidine] to its target enzyme dihydrofolate reductase has been investigated using a combination of 19F NMR spectroscopy and molecular mechanical calculations. 19F NMR reveals the presence of two different conformational states for the fluoronitropyrimethamine-Lactobacillus casei enzyme complex. MM2 molecular mechanical calculations predict restricted rotation about the C5-C1' bond of the ligand and this gives rise to two slowly interconverting rotamers which are an enantiomeric pair. The results of 19F NMR spectroscopy reveal that both these isomers bind to the enzyme, with different affinities. There is no detectable interconversion of the bound rotamers themselves on the NMR timescale. The effect of the addition of co-enzyme to the sample is to reverse the preference the enzyme has for each rotamer.

Folic Acid Antagonists↗
PubMed1988

500-MHz 1H-n.m.r. and conformational studies of fucosyloligosaccharides recognised by monoclonal antibodies with specificities related to Le(a), Le(b), and SSEA-1.

500-MHz 1H-n.m.r. spectroscopy has been used to examine several fucosylated oligosaccharides in studies to characterise carbohydrate antigenic determinants recognised by monoclonal antibodies. Reduction of the oligosaccharides to give additional variants for analysis showed that oligosaccharides having an alpha-L-fucosyl group linked to the reducing end residue have markedly different chemical shifts, and in some instances different antigenic activity, compared to their alditols. This information was incorporated into space filling molecular models of the oligosaccharides in order to predict the topography of atoms recognised by the antibody combining sites. These studies are an intermediate stage in the full characterisation of oligosaccharide conformation and molecular recognition by methods which accurately determine torsional angles and through-space internuclear distances.

Antibodies, Monoclonal↗
PubMed1988

Separation of human milk oligosaccharides by recycling chromatography. First isolation of lacto-N-neo-difucohexaose II and 3'-Galactosyllactose from this source.

Lacto-N-neo-difucohexaose II, beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-Glcp-NAc-(1----3)-beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-D-Glc, and 3'-galactosyllactose, beta-D-Galp-(1----3)-beta-D-Galp-(1----4)-D-Glc, were isolated for the first time from human milk by means of a recycling chromatography technique. Through this method, carried out mainly on columns of K+ ion-exchange resins and either Bio-Gel P-4 or TSK 40 W(S) gel filtration media, up to one gram of an oligosaccharide mixture could be handled and lacto-N-neo-difucohexaose II separated from isomeric lacto-N-difucohexaose I, alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc, and II, beta-D-Galp-(1----3)-[alpha L-Fucp-(1----4)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-D-Glc. This method also permitted resolution of isomeric mixtures of the trisaccharides 2'-fucosyllactose, alpha-L-Fucp-(1----2)-beta-D-Galp-(1----4)-D-Glc, and 3-fucosyllactose, beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-D-Glc, the tetrasaccharides lacto-N-tetraose, beta-D-Galp-(1----3)-beta-D-GlcpNac-(1----3)-beta-D-Galp-(1----4)-D-Glc, and lacto-N-neo-tetraose, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc, and the pentaoses lacto-N-fucopentaose I, alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc, II, beta-D-Galp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc, and III, beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc, which have proved difficult if not impossible to separate by other means. The isolation of these and other milk oligosaccharides is described herein. The 500-MHz 1H-n.m.r. spectra of lacto-N-neo-difucohexaose II and 3'-galactosyllactose, and their alditols, are recorded. 1H-n.m.r. data on some other milk oligosaccharides, both natural and reduced, are also given.

Carbohydrate Sequence↗
PubMed1988

500 MHz 1H n.m.r. of oligosaccharides of N-acetyl-lactosamine-type released from human erythrocyte glycopeptides by endo-beta-galactosidase.

500 MHz 1H n.m.r. spectroscopy has been used in structural studies of three linear and five branched oligosaccharides of N-acetyl-lactosamine-type that were released from desialylated blood group O erythrocyte glycopeptides by treatment with the endo-beta-galactosidase of Bacteroides fragilis followed by reduction. The following oligosaccharide alditols were characterized: (formula; see book)

Chemical Phenomena↗
PubMed1988

Dynamics of trimethoprim bound to dihydrofolate reductase.

The conformation of a small molecule in its binding site on a protein is a major factor in the specificity of the interaction between them. In this paper, we report the use of 1H and 13C NMR spectroscopy to study the fluctuations in conformation of the anti-bacterial drug trimethoprim when it is bound to its "target," dihydrofolate reductase. 13C relaxation measurements reveal dihedral angle changes of +/- 25 degrees to +/- 35 degrees on the subnanosecond time scale, while 13C line-shape analysis demonstrates dihedral angle changes of at least +/- 65 degrees on the millisecond time scale. 1H NMR shows that a specific hydrogen bond between the inhibitor and enzyme, which is believed to make an important contribution to binding, makes and breaks rapidly at room temperature.

Binding Sites↗
PubMed1987

Structural comparisons of complexes of methotrexate analogues with Lactobacillus casei dihydrofolate reductase by two-dimensional 1H NMR at 500 MHz.

We have used two-dimensional (2D) NMR methods to examine complexes of Lactobacillus casei dihydrofolate reductase and methotrexate (MTX) analogues having structural modifications of the benzoyl ring [the 3',5'-difluoro and 3',5'-dichloro analogues (II and III)] and also the glutamic acid moiety [the alpha- and gamma-monoamides (IV and V)]. Assignments of the 1H signals in the spectra of the various complexes were made by comparison of their 2D spectra with those of complexes containing methotrexate where we have previously assigned resonances from 32 of the 162 amino acid residues. In the complexes formed with the dihalomethotrexate analogues, the glutamic acid and pteridine ring moieties were shown to bind to the enzyme in a manner similar to that found in the methotrexate-enzyme complex. Perturbations in 1H chemical shifts of protons in Phe-49, Leu-54, and Leu-27 and the methotrexate H7 and NMe protons were observed in the different complexes and were accounted for by changes in orientation of the benzoyl ring in the various complexes (15 degrees and 25 degrees in the difluoro- and dichloromethotrexate complexes, respectively). Binding of oxidized or reduced coenzyme (NADP+ or NADPH) to the binary complexes did not result in different shifts for Leu-27, Leu-54, or Leu-19 protons, and thus, the orientation of the benzoyl ring of the methotrexate analogues is not perturbed greatly by the presence of either oxidized or reduced coenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Folic Acid Antagonists↗
PubMed1987

15N and 1H NMR evidence for multiple conformations of the complex of dihydrofolate reductase with its substrate, folate.

The binding of folate to Lactobacillus casei dihydrofolate reductase in the presence and absence of NADP+ has been studied by 15N NMR, using [5-15N]folate. In the presence of NADP+, three separate signals were observed for the single 15N atom, in agreement with our earlier evidence from 1H and 13C NMR for multiple conformations of this complex [(1982) Biochemistry 21, 5831-5838]. The 15N spectra of the binary enzyme-folate complex provide evidence for the first time that this complex also exists in at least two conformational states. This is confirmed by the observation of two separate resonances for the 7-proton of bound folate, located by two-dimensional exchange spectroscopy.

Bacterial Proteins↗
PubMed1986

Ricin-resistant mutants of baby-hamster-kidney cells deficient in alpha-mannosidase-II-catalyzed processing of asparagine-linked oligosaccharides.

Previous work has shown that two ricin-resistant mutants of baby hamster kidney (BHK) cells, RicR15 and RicR19, synthesize only hybrid and oligomannose-type asparagine-linked oligosaccharides [Hughes, R. C. and Mills, G. (1985) Biochem. J. 226, 487-498]. In the present report glycopeptides were released from disrupted cells by exhaustive digestion with pronase, fractionated by chromatography on concanavalin-A--Sepharose, DEAE-Sephacel and lentil-lectin--Sepharose and characterized by 500-MHz 1H-NMR spectroscopy. The major hybrid structure identified in both cell lines contains five mannose residues and the sequence NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----2 linked to the alpha 1----3 arm mannose of the core pentasaccharide. Analysis of extracts of normal or mutant cells has shown in the mutants a deficiency in alpha-mannosidase activity measured with p-nitrophenyl alpha-mannoside. This activity is swainsonine-sensitive and exhibits a pH optimum at about 6-6.5. Assays using a specific substrate for alpha-mannosidase II, a terminal processing glycosidase in conversion of penta-mannose hybrid intermediates to complex N-glycans, reveals a reduced activity in RicR15 cells. Analysis of glycopeptides obtained from cells labelled with [3H]fucose or [3H]galactose revealed a small proportion of branched complex N-glycans of normal structure in mutant cells.

Animals↗
PubMed1986

Oligosaccharides obtained from a blood-group-Sd(a+) Tamm-Horsfall glycoprotein. An n.m.r. study.

Treatment of Tamm-Horsfall urinary glycoprotein with Bacteroides fragilis endo-beta-galactosidase over a range of enzyme concentrations, pH and temperature resulted in the release of a small but constant proportion of the terminal sugars, which indicates the presence in the glycoprotein of relatively few enzyme-susceptible -GlcNAc beta 1-3Gal beta 1-4GlcNAc- units. Three oligosaccharides were isolated from the enzyme digest and characterized as Gal beta 1-4GlcNAc beta 1-3Gal, NeuAc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4GlcNAc beta 1-3Gal by methylation analysis and exo-glycosidase digestion. The alditols of these oligosaccharides and related structures were examined by 500 MHz 1H-n.m.r. spectroscopy aided by spin-spin decoupling and two-dimensional correlated spectroscopy. An almost complete assignment of proton shifts was possible, and significant differences between the signals of some of the protons in the blood-group-Sda-active oligosaccharide III and literature values for the corresponding signals in the structurally related Cad-blood-group determinant are noted.

Blood Group Antigens↗
PubMed1986

1H-NMR studies at 500 MHz of a neutral disaccharide and sulphated di-, tetra-, hexa- and larger oligosaccharides obtained by endo-beta-galactosidase treatment of keratan sulphate.

In the preceding paper in this journal, the major oligosaccharides obtained by endo-beta-galactosidase digestion of bovine corneal keratan sulphate were identified as a neutral disaccharide, GlcNAc beta 1-3Gal, and sulphated di-, tetra-, hexa-, octa- and decasaccharides based on the sequence (-3/4GlcNAc beta 1-3Gal beta 1-)n having 1, 3, 5, 7 and 9 sulphate groups, respectively. In the present study, these oligosaccharides have been analysed by 500-MHz 1H-NMR spectroscopy using spin-decoupling and two-dimensional correlated spectroscopy experiments. The NMR data confirm the beta-configuration of all the interglycosidic linkages and are consistent with an alternating sequence of----4GlcNAc and----3Gal, a non-reducing-end N-acetylglucosamine residue and a reducing-end galactose residue. The NMR data have also established that a sulphate group is linked to the C6 position of all sugar residues except the reducing-end galactose as follows: (Formula: see text). The signals of the protons attached to the sulphated carbon atoms show marked downfield shifts (approximately 0.4 ppm from equivalent protons of non-sulphated carbon atoms), while the protons at C5 vicinal to sulphated atoms show a change of 0.1-0.2 ppm and other protons of the sulphated monosaccharides show smaller changes in chemical shift (0.01-0.1 ppm). The proton at C4 of the non-sulphated reducing-end galactose linked at C3 also shows a significant change in chemical shift (0.03 ppm).

Animals↗
PubMed1986

Trimethoprim binding to Lactobacillus casei dihydrofolate reductase: a 13C NMR study using selectively 13C-enriched trimethoprim.

We have measured the 13C chemical shifts for trimethoprim molecules selectively enriched with 13C at the 2-, 4-, 5-, 6-, and 7-positions and the p-OCH3 position in their complexes with Lactobacillus casei dihydrofolate reductase in the presence and absence of coenzyme analogues. The C2 carbon shifts indicate that the pyrimidine ring is protonated at N1 in all the complexes of trimethoprim with the enzyme and coenzymes and in each case the pyrimidine ring is binding in a similar way to that of the corresponding part of methotrexate in the enzyme-methotrexate complex. The C6 carbon of trimethoprim shows a large upfield shift in all complexes (3.51 to 4.70 ppm) but no shift in the complex of 2,4-diaminopyrimidine with the enzyme: these shifts probably arise from steric interactions between the C1' and C2' carbons and the H6 proton, which approach van der Waals contact in the folded conformation adopted by trimethoprim when bound to the enzyme. The large shift observed for C6 in all complexes indicates that the basic folded conformation is present in all of them. A comparison of the 13C shifts in the enzyme-trimethoprim-NADPH complex with those in the enzyme-trimethoprim binary complex shows substantial changes even for carbons such as C6 and p-OCH3 (0.46 and -0.36 ppm, respectively), which are remote from the coenzyme: these are caused by ligand-induced conformational changes that may involve displacement of the helix containing residues 42-49.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗