ENDOR study of vanadyl(2+)-imidazole complexes in frozen aqueous solution

Mulks, C. F.; Kirste, B.; Willigen, H. van

doi:10.1021/ja00386a011

Cited by 53 publications

(38 citation statements)

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“…The exchangeable proton we investigated had its maximum-observed coupling in the perpendicular direction and displayed a much smaller interaction value. Also, the carbon-bound protons in the imidazole complexes [28] did not match sufficiently with values obtained here, so that transfer of assignments was not readily made. However, the exchangeable proton interaction could be related to an Nz proton of histidine, again by comparison with the values of NO-hemoglobin [30].…”

Section: Discussioncontrasting

confidence: 58%

“…By attributing the ENDOR spectral features in the 1 -11 MHz region of the B-site complex to I4N interactions of a coordinated histidine, we note that the results differ from V 0 2 + -nitrogen coordination in imidazole model complexes [28], in which the interaction is significantly smaller. We suppose that V 0 2 + coordinates to histidine in a nonequatorial fashion, similar to what is known for the His-Nz interaction in NO-ligated hemoglobin and myoglobin [30].…”

Section: Discussionmentioning

confidence: 63%

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Visible, EPR and electron nuclear double‐resonance spectroscopic studies on the two metal‐binding sites of oxovanadium (IV)‐substituted D‐xylose isomerase

Bogumil

Hüttermann

Kappl

et al. 1991

European Journal of Biochemistry

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The two metal-binding sites of the D-XylOSe isomerase from Streptomyces rubiginosus were studied using VOz + as a sensor for the ligand environment. Titration of the tetrameric enzyme with VOZf, followed by EPR spectroscopy and inhibition studies, show that the first four V 0 2 + equivalents occupy, in analogy to Co2+, Cd2+ and PbZ+, the binding site B. The visible absorption data and the EPR parameters indicate that a nitrogen ligand is involved in the ligand sphere of the high-affinity B site. The low-affinity A site could be studied selectively by blocking the B site with visible and EPR-silent Cd2 ' . The visible data and EPR parameters for this site are consistent with a ligand environment composed of oxygen donors without nitrogen ligation. The nitrogen coordination in the high-affinity site could be demonstrated by electron nuclear double-resonance (ENDOR) studies of the 4V02+ enzyme, and was assigned to a histidine ligand. The I4N resonances are interpreted in terms of a quartet with a coupling value of 13.2 MHz. 'H-ENDOR coupling of 1.7 MHz, exchangeable in DzO, has been assigned to the N -H proton of the histidine. Additional proton ENDOR couplings, which are not exchangeable, are due to protons bound to the carbon atoms of the histidine. For the low-affinity binding site, a nitrogen coordination could be definitely excluded by the ENDOR measurements. Exchangeable 'H-ENDOR couplings observed in this sample were assigned to H 2 0 ligands in the vicinity of VOZf. The results closely relate to what is known from X-ray structure. However, the relative affinities for the two binding sites seem not to be the same for different bivalent cations.In mixed metal samples with four V 0 2 + and four Co2+ equivalents, the V 0 2 + is distributed between both binding sites. Small changes in the complex geometry of the A site, indicated by different EPR features, seem to occur if the B site is occupied by Co2+ or by Cd2+The intracellular enzyme D-xylose isomerase (XylI) from Streptomyces rubiginosus ATCC 21 132 catalyses in viva the reversible isomerisation of a-D-xylose to CX-D-XylUlOSe. It also converts a-D-glucose to %-D-frUCtOSe, a reaction widely used in industrial processes [l -31.The enzyme exists as a tetramer, composed of four identical subunits with a M , of 172420 as determined by ultraviolet laser desorption ionisation mass spectrometry [4, 51. The activity of the enzyme is dependent on the presence of bivalent cations [l-31. In the native enzyme, addition of Mg2+ is necessary for the catalytical function. Low activity is also observed in the presence of Co2+, Mn2+ and Fez+ [4, 61. Metal-binding studies of the Co2+-substituted enzyme reveal that each subunit contains two binding sites with different affinities as determined by visible spectroscopy and activity measurements. The high-affinity site, denoted as the B site, has a distorted octahedral coordination geometry, whereasCorreApondence to

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Section: Discussioncontrasting

confidence: 58%

Section: Discussionmentioning

confidence: 63%

Visible, EPR and electron nuclear double‐resonance spectroscopic studies on the two metal‐binding sites of oxovanadium (IV)‐substituted D‐xylose isomerase

Bogumil

Hüttermann

Kappl

et al. 1991

European Journal of Biochemistry

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“…The apparent inhibitory effect of VO 2ϩ can be abolished by substitution of NH 4 ϩ for K ϩ in the reaction mixture whereas the reaction in the presence of Mg 2ϩ or Mn 2ϩ is not influenced by addition of NH 4 ϩ . Because VO 2ϩ can be coordinated by amines and other nitrogen-donor ligands 3 (41)(42)(43), the high concentration of NH 4 ϩ is likely to cause coordination to VO 2ϩ , converting the system to be equivalent to that in the absence of added divalent metal ion. 4 Table II compares the results of our studies showing the influence of divalent metal ions under single turnover conditions for both intrinsic GTPase and kirromycin-stimulated GTPase activity in the absence of programmed ribosomes.…”

Section: Role Of Mg 2ϩ In Ef-tu Actionmentioning

confidence: 99%

Mg2+ Is Not Catalytically Required in the Intrinsic and Kirromycin-stimulated GTPase Action of Thermus thermophilus EF-Tu

Rütthard

Banerjee

Makinen

2001

Journal of Biological Chemistry

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The influence of divalent metal ions on the intrinsic and kirromycin-stimulated GTPase activity in the absence of programmed ribosomes and on nucleotide binding affinity of elongation factor Tu (EF-Tu) from Thermus thermophilus prepared as the nucleotide-and Mg 2؉ -free protein has been investigated. The GTPase superfamily of proteins, known more commonly as G-proteins, are ubiquitous in cellular systems and serve as key regulatory molecules catalyzing the hydrolysis of the ␤,␥-phosphate bond in GTP (1-4). For most G-proteins this process yields a tightly bound enzyme-product complex requiring the action of a nucleotide exchange factor to kinetically facilitate exchange of the GTP for GDP in the active site. Nucleotide binding and hydrolysis are key to regulating the activity of these enzymes, and both of these steps occur in the presence of the divalent metal ion Mg 2ϩ as the naturally occurring cofactor in the cell. However, in EF-Tu 1 function it has not been possible hitherto to establish whether there is a differential role of the divalent metal ion in nucleotide hydrolysis as opposed to nucleotide binding.In all G-proteins defined hitherto by x-ray crystallographic studies, the substrate analog guanosine 5Ј-(␤,␥-imido)-triphosphate is bound as a complex with Mg 2ϩ in which the divalent metal ion is coordinated to oxygen atoms from the ␤-phosphate and ␥-phosphate groups, and the guanine and divalent metal ion binding sites appear to be tightly coupled (5, 6). For ras (7), for elongation factor Tu from both thermophilic bacteria (8 -11) and Escherichia coli (12, 13), and for G␣-subunits of heterotrimeric G-proteins (14, 15), the dissociation constants for release of protein-bound nucleotide measured in the presence of Mg 2ϩ indicate differential binding affinities for GDP and GTP by at least an order of magnitude. Binding affinities for both nucleotides are often measured kinetically by ligand displacement because G-proteins are less stable when prepared in their nucleotide-free form. Because nucleotide separation is necessary for complete removal of Mg 2ϩ from the protein, the less stable, nucleotide-free form of G-proteins complicates efforts to determine whether the divalent metal ion has differential roles in nucleotide hydrolysis and nucleotide binding.In contrast to the structural instability of elongation factor Tu of mesophiles such as E. coli, the homologous thermostable protein in its nucleotide-free form is less prone to inactivation, and comparison of the kinetics of nucleotide binding shows it to be identical to EF-Tu isolated as the GDP-bound complex from the cytosol (11,16). This characteristic has allowed application of efficient biochemical purification methods for preparation of the nucleotide-free elongation factor from Thermus thermophilus (16,17) and Bacillus stearothermophilus (18, 19) for x-ray structure analysis of the complex formed with an inhibitor analog of GTP in the active site (5, 6). Isolation of nucleotidefree elongation factor Tu (17-20) and p21 ras (7) has also allowed comp...

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“…Table 1). Both the ribbon-wound and from an X-band experiment given in parentheses (18). Also wire-wound cavities have greater modulation penetration observed in the parallel spectrum (H z) is a hyperfine than the thin-silver-wall cavity (10 G) since opposing fields splitting of 6 MHz (6 MHz) due to protons of an axially from induced eddy currents are minimized in the spiral struccoordinated water molecule.…”

Section: Epoxy and Epoxy/quartz Formulationmentioning

confidence: 97%

“…tected in the X-band ENDOR experiment (18). Only the n 0 The power saturation curves of weak pitch in the various branch (n / Å n H / A/2; n 0 Å n H 0 A/2) is seen for the cavities are shown in Fig.…”

Section: Epoxy and Epoxy/quartz Formulationmentioning

confidence: 98%

Construction and Performance of Ribbon-Wound TE011Cylindrical EPR/ENDOR Cavities

Wang

Chasteen

1995

Journal of Magnetic Resonance, Series A

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ENDOR study of vanadyl(2+)-imidazole complexes in frozen aqueous solution

Cited by 53 publications

References 0 publications

Visible, EPR and electron nuclear double‐resonance spectroscopic studies on the two metal‐binding sites of oxovanadium (IV)‐substituted D‐xylose isomerase

Visible, EPR and electron nuclear double‐resonance spectroscopic studies on the two metal‐binding sites of oxovanadium (IV)‐substituted D‐xylose isomerase

Mg2+ Is Not Catalytically Required in the Intrinsic and Kirromycin-stimulated GTPase Action of Thermus thermophilus EF-Tu

Construction and Performance of Ribbon-Wound TE011Cylindrical EPR/ENDOR Cavities

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