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B J Gaffney

Publications and source records attributed to B J Gaffney.

At least 19 recordsLinked to original sources
PubMed1997

Structure and mechanism of lipoxygenases.

In mammals, lipoxygenases catalyze the formation of hydroperoxides as the first step in the biosynthesis of several inflammatory mediators. The substrate of this reaction, arachidonic acid, is the key precursor of two families of potent physiological effectors. It is the branch point between two central pathways: one, involving the enzyme cyclooxygenase, leads to the synthesis of prostaglandins and thromboxanes; the other, involving lipoxygenases, leads to the synthesis of leukotrienes and lipoxins, compounds that regulate important cellular responses in inflammation and immunity. While aspirin and other non-steroidal anti-inflammatory compounds are potent inhibitors of cyclooxygenase, no effective pharmacological inhibitor of lipoxygenase is presently available. Lipoxygenases are large non-heme, iron-containing enzymes that use molecular oxygen for the diooxygenation of arachidonic acid to form hydroperoxides, the first step in the biosynthetic pathways leading to leukotrienes and lipoxins. Because of the importance of these compounds, lipoxygenases have been the subject of extensive study: from detailed kinetic measurements to cloning, expression, and site-directed mutagenesis. The sequences of over 50 lipoxygenases have been reported. In addition, the structure of soybean lipoxygenase-1, determined by X-ray diffraction methods, has recently been reported. The structure revealed that the 839 amino acids in the protein are organized in two domains: a beta-sheet N-terminal domain and a large, mostly helical C-terminal domain. The iron is present in the C-terminal domain facing two internal cavities that are probably the conduits through which the fatty acid and molecular oxygen gain access to the metal. Models of the mammalian lipoxygenases based on the soybean structure provide clues about the structural determinants of the positional specificity of the enzyme, and can be used as targets for the design of more effective inhibitors.

Animals↗
PubMed1997

Interdoublet transitions in S = 5/2 protein systems.

Beginning with known parameters that characterize the EMR spectra of several proteins containing high-spin ferric iron, the information content of the spectra has been examined by simulations that cover a range of magnetic fields and frequencies. Transitions between levels that are not Kramers doublet levels are particularly interesting when the applied frequency is approximately two to three times the value of the zero-field splitting parameter, D. In these cases, transitions at very low magnetic fields correspond to portions of interdoublet transitions that are well separated from all other transitions. The magnetic field is aligned at angles between the molecular principal axes for the portion of the molecules giving rise to the low-field interdoublet transitions. This provides an opportunity for unique angle-selection experiments.

Electron Spin Resonance Spectroscopy↗
PubMed1996

Structure conservation in lipoxygenases: structural analysis of soybean lipoxygenase-1 and modeling of human lipoxygenases.

Lipoxygenases are a class of non-heme iron dioxygenases which catalyze the hydroperoxidation of fatty acids for the biosynthesis of leukotrienes and lipoxins. The structure of the 839-residue soybean lipoxygenase-1 was used as a template to model human 5-, 12-, and 15-lipoxygenases. A distance-based algorithm for placing side chains in a low homology environment (only the four iron ligands were fixed during side chain placement) was devised. Twenty-six of the 56 conserved lipoxygenase residues were grouped in four distinct regions of the enzyme. These regions were analyzed to discern whether the side chain interactions could be duplicated in the models or whether alternate conformers should be considered. The effects of site directed mutagenesis variants were rationalized using the models of the human lipoxygenases. In particular, variants which shifted positional specificity between 12- and 15-lipoxygenase activity were analyzed. Analysis of active site residues produced a model which accounts for observed lipoxygenase positional specificity and stereospecificity.

Algorithms↗
PubMed1996

Lipoxygenases: structural principles and spectroscopy.

Lipoxygenases catalyze the formation of fatty acid hydroperoxides, products used in further biochemical reactions leading to normal and pathological cell functions. X-ray structure analysis and spectroscopy have been applied to elucidate the mechanism of lipoxygenases. Two X-ray structures of soybean lipoxygenase-1 reveal the side chains of three histidines and the COO- of the carboxy terminus as ligands to the catalytically important iron atom. The enzyme contains a novel three-turn pi-helix near the iron center. Spectroscopic studies, including electron magnetic resonance, X-ray absorption spectroscopy, infrared circular dichroism, and magnetic circular dichroism, have been applied to compare lipoxygenases from varied sources and with different substrate positional specificity.

Animals↗
PubMed1993

The three-dimensional structure of an arachidonic acid 15-lipoxygenase.

In mammals, the hydroperoxidation of arachidonic acid by lipoxygenases leads to the formation of leukotrienes and lipoxins, compounds that mediate inflammatory responses. Lipoxygenases are dioxygenases that contain a nonheme iron and are present in many animal cells. Soybean lipoxygenase-1 is a single-chain, 839-residue protein closely related to mammalian lipoxygenases. The structure of soybean lipoxygenase-1 solved to 2.6 angstrom resolution shows that the enzyme has two domains: a 146-residue beta barrel and a 693-residue helical bundle. The iron atom is in the center of the larger domain and is coordinated by three histidines and the COO- of the carboxyl terminus. The coordination geometry is nonregular and appears to be a distorted octahedron in which two adjacent positions are not occupied by ligands. Two cavities, in the shapes of a bent cylinder and a frustum, connect the unoccupied positions to the surface of the enzyme. The iron, with two adjacent and unoccupied positions, is poised to interact with the 1,4-diene system of the substrate and with molecular oxygen during catalysis.

Amino Acid Sequence↗
PubMed1993

Access of ligands to the ferric center in lipoxygenase-1.

A form of ferric lipoxygenase-1 has been isolated that gives an EPR spectrum that is dominated by a species of intermediate rhombicity (E/D = 0.065). This species is obtained in the presence of a number of buffers of high concentration and in the absence of fatty acid byproducts of the iron oxidation. The species is unstable over a period of one day with respect to symmetry of the iron. The EPR lineshapes of the unstable species are highly sensitive to the anionic composition of the buffer and to the addition of neutral ligands. These results suggest that newly formed ferric lipoxygenase has weak affinity for a number of ligands. Affinity of charged ligands for the iron center may provide a mechanism for charge compensation as the iron center alternates between ferric and ferrous in the catalytic cycle. We use spectral simulation to evaluate quantitatively the interaction of the ferric center with ligands and also show that a transition in the middle Kramers doublet makes a significant contribution to the EPR spectrum of the more rhombic species.

Binding Sites↗
PubMed1991

Effect of the synergistic anion on electron paramagnetic resonance spectra of iron-transferrin anion complexes is consistent with bidentate binding of the anion.

Continuous wave (cw) X-band EPR spectra at approximately 90 K were obtained for iron-transferrin-anion complexes with 18 anions. Each anion had a carboxylate group and at least one other polar moiety. As the second polar group was varied from hydroxyl to carbonyl to amine to carboxylate, the EPR spectra changed from a dominant signal at g' approximately 4.3 with a second smaller peak at g' approximately 9 to a broad signal with intensity between g' approximately 5 and 7. Computer simulation indicated that the changes in the EPR spectra were due to changes in the zero field splitting parameter ratio, E/D, from approximately 1/3 for carbonate anion to approximately 0.04 for malonate anion. Observation of iron-13C coupling in the electron spin echo envelope modulation (ESEEM) for iron transferrin [1-13C]pyruvate indicated that the carboxylate group was bound to the iron. It is proposed that all of the anions behave as bidentate ligands, with coordination to the iron through both the carboxylate and proximal groups, and the carboxyl group serves as a bridge between the iron and a positively charged group on the protein.

Anions↗
PubMed1990

Crystallization and preliminary x-ray analysis of soybean lipoxygenase-1, a non-heme iron-containing dioxygenase.

Crystals of lipoxygenase-1 from soybeans have been grown by the method of vapor diffusion in the presence of sodium formate, ammonium acetate, and lithium chloride at pH 7.0. This enzyme contains a non-heme iron and is closely related to a human lipoxygenase found in leukocytes that participates in the biosynthesis of leukotrienes and lipoxins. The crystals are monoclinic space group C2 with cell dimensions of a = 183.8 A, b = 123.2 A, c = 94.3 A and beta = 102.9 degrees. They diffract beyond 2.7 A, are stable for several days in the x-ray beam, and appear to be suitable for x-ray diffraction studies.

Chromatography, High Pressure Liquid↗
PubMed1987

Determination of relative spin concentration in some high-spin ferric proteins using E/D-distribution in electron paramagnetic resonance simulations.

Lineshape simulations are presented for the multiple, overlapping X-band electron paramagnetic resonance (EPR) spectra in two non-heme, high-spin iron proteins: phenylalanine hydroxylase (PAH) and diferric transferrin. The aim of the calculations is to determine the fraction of iron contributing to each of the sites visible by EPR. The simulations are limited to the experimentally accessible transitions occurring at g-values greater than 1.7. In both PAH and transferrin, at least one of the iron sites is characterized by the ratio of zero-field splitting parameters, E/D, near 1/3 and a broad, asymmetric lineshape. A distribution in E/D-values is used in the simulations to account for this breadth and asymmetry. To test the E/D-distribution model, experimental X-band spectra of diferric transferrin at several salt concentrations are fit by simulation. In this test, first the low-field features arising from transitions between the lowest Kramers doublet levels are simulated using E/D-distributions for two sites. Second, parameters that provide a good fit for the lowest doublet transitions are shown also to fit the resonance near an effective g-value of 4.3 from the middle Kramers doublet transition. When applied to spectra of PAH in the resting state, the E/D-distribution approach accounts for the intensity of one of the two major species of iron. The other species is characterized by E/D = 0.032, and the spectrum of this portion of the resting enzyme may be simulated using a frequency-swept Gaussian lineshape. Spectra for the enzyme in an inhibitor-saturated state are also simulated. The simulations are consistent with previous biochemical studies that indicate that only the E/D = 0.032 form of iron participates in catalysis.

Binding Sites↗
PubMed1986

Characterization of phenylalanine hydroxylase.

Iron can be bound to phenylalanine hydroxylase (PAH) in two environments. The assignment of the electron paramagnetic resonance spectrum of PAH to two, overlapping high-spin ferric signals is confirmed by computer simulation. Both environments are shown to be populated in the crude enzyme. Reconstitution of the apoenzyme demonstrated that the two iron environments are not interconvertible. Oxygen consumption during PAH reduction by tetrahydropterin in the absence of phenylalanine but not in its presence explains the different reduction stoichiometries (tetrahydropterin:enzyme) that have been observed.

Animals↗