The Experts below are selected from a list of 21372 Experts worldwide ranked by ideXlab platform
Robert Callender - One of the best experts on this subject based on the ideXlab platform.
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the assignment of downfield proton resonances in an Enzyme Inhibitor Complex using time dependent saturation transferred noes
2004Co-Authors: Hua Deng, Linda C Kurz, Sean M Cahill, Robert CallenderAbstract:We have used time-dependent saturation transferred NOE (STNOE) measurements to assign two downfield resonances in the proton spectrum of an adenosine deaminase−purine riboside mixture. Our results show that this method can be used to detect structural changes that occur upon Inhibitor binding to the Enzyme and to determine which protons of the bound Inhibitor are strongly hydrogen bonded in the Complex.
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the assignment of downfield proton resonances in an Enzyme Inhibitor Complex using time dependent saturation transferred noes
2004Co-Authors: Hua Deng, Linda C Kurz, Sean M Cahill, Robert CallenderAbstract:We have used time-dependent saturation transferred NOE (STNOE) measurements to assign two downfield resonances in the proton spectrum of an adenosine deaminase−purine riboside mixture. Our results show that this method can be used to detect structural changes that occur upon Inhibitor binding to the Enzyme and to determine which protons of the bound Inhibitor are strongly hydrogen bonded in the Complex.
Koji Takio - One of the best experts on this subject based on the ideXlab platform.
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characterization of the interface structure of Enzyme Inhibitor Complex by using hydrogen deuterium exchange and electrospray ionization fourier transform ion cyclotron resonance mass spectrometry
2000Co-Authors: Satoko Akashi, Koji TakioAbstract:We investigated the interaction between a thiol protease Inhibitor, cystatin, and its target Enzyme, papain, by hydrogen-deuterium (H/D) exchange in conjunction with successive analysis by collision-induced dissociation (CID) in an rf-only hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium incorporation into backbone amide hydrogens of cystatin was analyzed at different time points in the presence or absence of papain, examining the mass of each fragment produced by hexapole-CID. In the absence of papain, amide hydrogens in short amino-terminal fragments, such as b10(2+) and b12(2+), were highly deuterated within 1 min. Although fewer fragments were observed for the cystatin-papain Complex in the hexapole-CID spectra, significant reductions in initial deuterium content were recognized throughout the sequence of cystatin. This suggests that Complex formation restricted the flexibility of the whole cystatin molecule. Detailed analyses revealed that a marked reduction in deuterium content in the region of residues 1-10 persisted for hours, suggesting that the flexible N-terminal region was tightly fixed in the binding pocket with hydrogen bonds. Our results are consistent with those of previous studies on the structure and inhibition mechanism of cystatin. We demonstrated here that Enzyme-Inhibitor interactions can be characterized by H/D exchange in combination with CID in a hexapole ion guide using ESI-FTICR MS rapidly and using only a small amount of sample.
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characterization of the interface structure of Enzyme Inhibitor Complex by using hydrogen deuterium exchange and electrospray ionization fourier transform ion cyclotron resonance mass spectrometry
2000Co-Authors: Satoko Akashi, Koji TakioAbstract:We investigated the interaction between a thiol protease Inhibitor, cystatin, and its target Enzyme, papain, by hydrogen-deuterium (H/D) exchange in conjunction with successive analysis by collision-induced dissociation (CID) in an rf-only hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium incorporation into backbone amide hydrogens of cystatin was analyzed at different time points in the presence or absence of papain, examining the mass of each fragment produced by hexapole-CID. In the absence of papain, amide hydrogens in short amino-terminal fragments, such as b10(2+) and b12(2+), were highly deuterated within 1 min. Although fewer fragments were observed for the cystatin-papain Complex in the hexapole-CID spectra, significant reductions in initial deuterium content were recognized throughout the sequence of cystatin. This suggests that Complex formation restricted the flexibility of the whole cystatin molecule. Detailed analyses revealed that a marked reduction in deuterium content in the region of residues 1-10 persisted for hours, suggesting that the flexible N-terminal region was tightly fixed in the binding pocket with hydrogen bonds. Our results are consistent with those of previous studies on the structure and inhibition mechanism of cystatin. We demonstrated here that Enzyme-Inhibitor interactions can be characterized by H/D exchange in combination with CID in a hexapole ion guide using ESI-FTICR MS rapidly and using only a small amount of sample.
Hua Deng - One of the best experts on this subject based on the ideXlab platform.
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the assignment of downfield proton resonances in an Enzyme Inhibitor Complex using time dependent saturation transferred noes
2004Co-Authors: Hua Deng, Linda C Kurz, Sean M Cahill, Robert CallenderAbstract:We have used time-dependent saturation transferred NOE (STNOE) measurements to assign two downfield resonances in the proton spectrum of an adenosine deaminase−purine riboside mixture. Our results show that this method can be used to detect structural changes that occur upon Inhibitor binding to the Enzyme and to determine which protons of the bound Inhibitor are strongly hydrogen bonded in the Complex.
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the assignment of downfield proton resonances in an Enzyme Inhibitor Complex using time dependent saturation transferred noes
2004Co-Authors: Hua Deng, Linda C Kurz, Sean M Cahill, Robert CallenderAbstract:We have used time-dependent saturation transferred NOE (STNOE) measurements to assign two downfield resonances in the proton spectrum of an adenosine deaminase−purine riboside mixture. Our results show that this method can be used to detect structural changes that occur upon Inhibitor binding to the Enzyme and to determine which protons of the bound Inhibitor are strongly hydrogen bonded in the Complex.
Michael A Bernstein - One of the best experts on this subject based on the ideXlab platform.
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nmr structural studies of the tight Complex between a trifluoromethyl ketone Inhibitor and the 85 kda human phospholipase a2
1993Co-Authors: Laird A Trimble, Ian P Street, Helene Perrier, Nathalie Tremblay, Philip K Weech, Michael A BernsteinAbstract:Arachidonyl trifluoromethyl ketone (AACOCF 3 ) is a slow- and tight-binding Inhibitor of the human cytosolic phospholipase A 2 (cPLA 2 ) [Street et al. (1993) Biochemistry 32, 5935]. 19 F and 13 C NMR experiments have been carried out to elucidate the structure of the cPLA 2 .AACOCF 3 Complex. One mole of AACOCF 3 per mole of Enzyme is tightly bound in the active site while excess molar equivalents of the Inhibitor associate loosely and nonspecifically with hydrophobic regions of the protein. Incubation of the' cPLA 2 .AACOCF 3 Complex with a 10-fold molar excess of a structurally related Inhibitor allows the slow dissociation of the Enzyme-Inhibitor Complex to be followed with 19 F NMR
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nmr structural studies of the tight Complex between a trifluoromethyl ketone Inhibitor and the 85 kda human phospholipase a2
1993Co-Authors: Laird A Trimble, Ian P Street, Helene Perrier, Nathalie Tremblay, Philip K Weech, Michael A BernsteinAbstract:: Arachidonyl trifluoromethyl ketone (AACOCF3) is a slow- and tight-binding Inhibitor of the human cytosolic phospholipase A2 (cPLA2) [Street et al. (1993) Biochemistry 32, 5935]. 19F and 13C NMR experiments have been carried out to elucidate the structure of the cPLA2.AACOCF3 Complex. One mole of AACOCF3 per mole of Enzyme is tightly bound in the active site while excess molar equivalents of the Inhibitor associate loosely and nonspecifically with hydrophobic regions of the protein. Incubation of the cPLA2.AACOCF3 Complex with a 10-fold molar excess of a structurally related Inhibitor allows the slow dissociation of the Enzyme-Inhibitor Complex to be followed with 19F NMR. These results establish that the bound Inhibitor is in slow exchange with the free ligand and that inhibition of the cPLA2 by AACOCF3 is not due to irreversible modification of the protein. AACOCF3 labeled with 13C at the carbonyl position was used to determine the nature of the bound Inhibitor species. A comparison of the 13C NMR chemical shift value obtained from labeled Enzyme-Inhibitor Complex (delta c 101.0 ppm) with the chemical shift values obtained from model compounds suggests that the Enzyme-bound Inhibitor species is a charged hemiketal. These results are very similar to those obtained previously with alpha-chymotrypsin and a peptidyl trifluoromethyl ketone Inhibitor [Liang, T.-C., & Abeles, R. H. (1987) Biochemistry 26, 7603] and, by analogy with the serine proteases, a structural model for the cPLA2.AACOCF3 Complex is proposed.
Vasanti V Deshpande - One of the best experts on this subject based on the ideXlab platform.
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slow tight binding inhibition of proteinase k by a proteinaceous Inhibitor conformational alterations responsible for conferring irreversibility to the Enzyme Inhibitor Complex
2003Co-Authors: Jui Pandhare, Chandravanu Dash, Vasanti V DeshpandeAbstract:Abstract The kinetics of slow onset inhibition of Proteinase K by a proteinaceous alkaline protease Inhibitor (API) from a Streptomyces sp. is presented. The kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 ± 0.5 × 10–5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible Enzyme-Inhibitor Complex (EI) with a Ki value 5.2 ± 0.6 × 10–6 m. The EI Complex isomerized to a stable Complex (EI*) in the second step because of Inhibitor-induced conformational changes, with a rate constant k5 (9.2 ± 1 × 10–3 s–1). The rate of dissociation of EI* (k6) was slower (4.5 ± 0.5 × 10–5 s–1) indicating the tight binding nature of the Inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 ± 0.3 × 10–7 m. Time-dependent dissociation of EI* revealed that the Complex failed to dissociate after a time point and formed a conformationally altered, irreversible Complex EI**. These conformational states of Enzyme-Inhibitor Complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the Enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locked EI** Complex. API binds to the active site of the Enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the catalytic triad and other residues involved in catalysis.
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slow tight binding inhibition of proteinase k by a proteinaceous Inhibitor conformational alterations responsible for conferring irreversibility to the Enzyme Inhibitor Complex
2003Co-Authors: Jui Pandhare, Chandravanu Dash, Vasanti V DeshpandeAbstract:Abstract The kinetics of slow onset inhibition of Proteinase K by a proteinaceous alkaline protease Inhibitor (API) from a Streptomyces sp. is presented. The kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 ± 0.5 × 10–5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible Enzyme-Inhibitor Complex (EI) with a Ki value 5.2 ± 0.6 × 10–6 m. The EI Complex isomerized to a stable Complex (EI*) in the second step because of Inhibitor-induced conformational changes, with a rate constant k5 (9.2 ± 1 × 10–3 s–1). The rate of dissociation of EI* (k6) was slower (4.5 ± 0.5 × 10–5 s–1) indicating the tight binding nature of the Inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 ± 0.3 × 10–7 m. Time-dependent dissociation of EI* revealed that the Complex failed to dissociate after a time point and formed a conformationally altered, irreversible Complex EI**. These conformational states of Enzyme-Inhibitor Complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the Enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locked EI** Complex. API binds to the active site of the Enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the catalytic triad and other residues involved in catalysis.