An Arginine Residue Is Essential for Stretching and Binding of the Substrate on UDP-glucose-4-epimerase from Escherichia coli

Bhattacharyya, Uma; Dhar, Gautam; Bhaduri, Amar

doi:10.1074/jbc.274.21.14573

Cited by 6 publications

(4 citation statements)

References 32 publications

(27 reference statements)

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“…Marked fluorescence enhancements have been observed for a more extended configuration induced by the nucleotide-binding site of a protein, due to hindrance of this chromophore-base quenching interaction. Several proteins have been studied in this way, such as RNA polymerase (14), UDP-glucose 4-epimerase (15), and a large number of dehydrogenases using NAD or etheno-NAD as probes (16). Draganescu et al (17) have described the binding of BODIPY FL GTP-␥-S and other nonfluorescent ␥-phosphatelinked analogs to Fhit, a member of the histidine triad superfamily of nucleotide-binding proteins involved in tumor suppression.…”

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confidence: 99%

Spontaneous nucleotide exchange in low molecular weight GTPases by fluorescently labeled γ-phosphate-linked GTP analogs

Korlach

Baird

Heikal

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Regulated guanosine nucleotide exchange and hydrolysis constitute the fundamental activities of low molecular weight GTPases. We show that three guanosine 5′-triphosphate analogs with BODIPY fluorophores coupled via the gamma phosphate bind to the GTPases Cdc42, Rac1, RhoA, and Ras and displace guanosine 5′-diphosphate with high intrinsic exchange rates in the presence of Mg 2+ ions, thereby acting as synthetic, low molecular weight guanine nucleotide exchange factors. The accompanying large fluorescence enhancements (as high as 12-fold), caused by a reduction in guanine quenching of the environmentally sensitive BODIPY dye fluorescence on protein binding, allow for real-time monitoring of this spontaneous nucleotide exchange in the visible spectrum with high signal-to-noise ratios. Binding affinities increased with longer aliphatic linkers connecting the nucleotide and BODIPY fluorophore and were in the 10–100 nM range. Steady-state and time-resolved fluorescence spectroscopy showed an inverse relationship between linker length and fluorescence enhancement factors and differences in protein-bound fluorophore mobilities, providing optimization criteria for future applications of such compounds as efficient elicitors and reporters of nucleotide exchange. EDTA markedly enhanced nucleotide exchange, enabling rapid loading of GTPases with these probes. Differences in active site geometries, in the absence of Mg 2+ , caused qualitatively different reporting of the bound state by the different analogs. The BODIPY analogs also prevented the interaction of Cdc42 with p21 activated kinase. Together, these results validate the use of these analogs as valuable tools for studying GTPase functions and for developing potent synthetic nucleotide exchange factors for this important class of signaling molecules.

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confidence: 99%

Spontaneous nucleotide exchange in low molecular weight GTPases by fluorescently labeled γ-phosphate-linked GTP analogs

Korlach

Baird

Heikal

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Folding studies can also be facilitated by using these probes to monitor the generation of ligand binding site during the folding process. In the following paper usefulness of UDPAmNS as a quenched fluorophore will be demonstrated taking UDPglucose-4-epimerase from Escherichia coli as the target enzyme (32).…”

Section: Discussionmentioning

confidence: 99%

“…ATPAmNS, GTPAmNS, and PPAmNS failed to show any significant enhancement under identical conditions. Most of the subsequent spectroscopic work reported here was carried out with UDPAmNS, because this molecule showed maximum enhancement of fluorescence and was also used as the probe for our subsequent study with UDPgalactose-4-epimerase, as reported in the following paper (32).…”

Section: H Nmr Spectroscopy-thementioning

confidence: 99%

Synthesis and Characterization of Stacked and Quenched Uridine Nucleotide Fluorophores

Dhar

Bhaduri

1999

Journal of Biological Chemistry

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Since the seminal work of Weber and Laurence (1) with 1-anilinonaphthalene-8-sulfonic acid, extrinsic fluorescent probes have been extensively used to monitor various aspects of protein-ligand or enzyme-substrate interactions. Among others, these probes have been used (i) to establish the degree of polarity or hydrophobicity of a particular region of a protein, (ii) to measure the distance between groups on protein surface, (iii) to measure the extent of flexibility of protein in solution, (iv) to measure the rate of very rapid conformational transitions, and (v) to measure kinetic constants of interaction between protein and ligand (2). To facilitate these studies, a variety of derivatized fluorescent ligands or substrate analogs have been synthesized over the years without any serious attention being given to their solution conformations or their transitions to new conformations on interaction with the target proteins. Such conformational transitions are often crucial steps in biological interactions as typically exemplified by NAD, the common cofactor for a very large number of dehydrogenases. The molecule exhibits reversible stacking between the adenine and pyridine moiety with both the open and the closed forms in rapid interconversionary equilibrium in aqueous solutions (3, 4). During catalysis, NAD takes a totally extended conformation on the enzyme surface; the conserved tertiary structure of the pyridine nucleotide binding site being a very characteristic feature of all these oxidoreductases (5).Etheno-ATP was originally synthesized as a fluorescent analog of ATP. It had a high quantum yield and a long fluorescence lifetime and could be used to follow ATP interactions primarily by polarization studies (6 -8). In contrast, as in case of NAD, significant population of etheno-NAD was in a folded conformation in aqueous solution as a result of aromatic interactions between the pyridine and the modified adenine moiety, leading to dynamic collisional quenching of fluorescence and short fluorescence life-time (9). This stacked and quenched fluorophore was brilliantly used to establish negative co-operativity for glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle for the binding of the tetrameric apoenzyme to the coenzyme. The conformational transition of etheno-NAD from folded to stretched conformation as reflected by its enhanced fluorescence on interaction with the target protein was the monitoring parameter for this purpose (10). Although stacked fluorophore with quenched fluorescence can be of immense use in protein-ligand binding studies, as is exemplified by etheno-NAD, it is surprising to note that no deliberate effort has so far been made to design such compounds taking advantage of the potential aromatic interaction between the attached fluorophore and a suitable moiety of the desired biomolecule.The interaction between aromatic rings is of wide chemical and biological interest, because it plays important roles in vital biological processes, such as stabilization of protein and nucleic acid s...

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“…Compared with the ϳ15-fold increase in fluorescence of NADH bound to deoxyhypusine synthase, the increase in peak intensity in 90% tetrahydrofuran was only 3-fold that of the same concentration of NADH in buffer, suggesting that there may be additional factors affecting the enhancement of fluorescence emission. One is the relief of both static and collisional quenching between the adenine and dihydronicotinamide moieties that occurs in the "closed" conformation of NADH in aqueous solution, which has been cited by Weber and others (20,21,35,36). When bound to an enzyme, the NADH generally assumes a more "open" conformation, increasing the distance between the two ring systems and thus increasing the fluorescence emission (20).…”

Section: Figmentioning

confidence: 99%

Deoxyhypusine Synthase Generates and Uses Bound NADH in a Transient Hydride Transfer Mechanism

Wolff¹,

Wolff²,

Park³

2000

Journal of Biological Chemistry

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Deoxyhypusine is a modified lysine residue. It is formed posttranslationally in the precursor of eukaryotic initiation factor 5A (eIF5A) by deoxyhypusine synthase, employing spermidine as a butylamine donor. In the initial step of this reaction, deoxyhypusine synthase catalyzes the production of NADH through dehydrogenation of spermidine. Fluorescence measurements of this reaction revealed a ؊22-nm blue shift in the emission peak of NADH and a ϳ15-fold increase in peak intensity, characteristics of tightly bound NADH that were not seen by simply mixing NADH and enzyme. The fluorescent properties of the bound NADH can be ascribed to a hydrophobic environment and a rigidly held, open conformation of NADH, features in accord with the known crystal structure of the enzyme. Considerable fluorescence resonance energy transfer from tryptophan 327 in the active site to the dihydronicotinamide ring of NADH was seen. Upon addition of the eIF5A precursor, utilization of the enzyme-bound NADH for reduction of the eIF5A-imine intermediate to deoxyhypusine was reflected by a rapid decrease in the NADH fluorescence, indicating a transient hydride transfer mechanism as an integral part of the reaction. The number of NADH molecules bound approached four/enzyme tetramer; not all of the bound NADH was available for reduction of the eIF5A-imine intermediate.

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An Arginine Residue Is Essential for Stretching and Binding of the Substrate on UDP-glucose-4-epimerase from Escherichia coli

Cited by 6 publications

References 32 publications

Spontaneous nucleotide exchange in low molecular weight GTPases by fluorescently labeled γ-phosphate-linked GTP analogs

Spontaneous nucleotide exchange in low molecular weight GTPases by fluorescently labeled γ-phosphate-linked GTP analogs

Synthesis and Characterization of Stacked and Quenched Uridine Nucleotide Fluorophores

Deoxyhypusine Synthase Generates and Uses Bound NADH in a Transient Hydride Transfer Mechanism

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