Vibrational studies of phosphoryl transfer enzymes: ras-p21(*)magnesium-GTP and Myosin S1(*)magnesium-ADP-vanadate.
Item
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Title
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Vibrational studies of phosphoryl transfer enzymes: ras-p21(*)magnesium-GTP and Myosin S1(*)magnesium-ADP-vanadate.
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Identifier
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AAI9917711
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identifier
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9917711
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Creator
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Wang, Jianghua.
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Contributor
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Adviser: Robert H. Callender
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Date
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1999
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Language
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English
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Publisher
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City University of New York.
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Subject
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Physics, Molecular | Chemistry, Biochemistry
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Abstract
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We have measured the Raman spectra of monophosphate compounds in aqueous solution. The measured frequencies were correlated with P••O valence bond order by using a modification of the Hardcastle-Wachs procedure. The P••O bond order and bond length in phosphates can be determined from vibrational spectra by using the derived bond order/stretching frequency correlation and the bond length/bond order correlation of Brown and Wu.;The Raman and infrared spectra of guanosine 5'-diphosphate (GDP) and guanosine 5'-triphosphate (GTP) in aqueous solution were also examined. Frequency shifts were observed as Mg2+ complexes with GDP and GTP in aqueous solution. These results suggested that Mg2+ binds to GDP in a bidentate manner to the alpha,beta P••O bonds and in a tridentate manner to the alpha,beta and gamma P••O bonds of Mg•GTP . We have analyzed the previously obtained isotope edited Raman difference spectra of 1:1 complexes of Mg•GDP and Mg•GTP in ras-p21. Frequency changes of the phosphate groups were observed when Mg•GDP , Mg•GTP bind to the protein. Employing both the previous empirical relationships between bond orders/lengths and frequencies as well as vibrational analysis from ab initio calculations, the spectral changes can be explained by the change of the Mg2+ binding sites and hydrogen-bonding. Implications of these structural results for the reaction mechanism of GTP hydrolysis catalyzed by the GTPase are discussed.;We have analyzed previously obtained isotope edited Raman difference spectra of the non-bridging V••O bonds of vanadates, both in solution, and when bound to the myosin S1•MgADP complex. By use of ab initio calculations on a model of the vanadate binding site in myosin, the angles between the non-bridging V••O bonds and between these bonds and the apical bonds in the myosin S1•MgADP -Vi complex were determined. The summed bond order of the two apical bonds between the attacking and leaving group oxygens with the central vanadium ion in the S1•MgADP -Vi complex was found to increase only slightly compared with the bond order of the ester V-O bond of a monoester vanadate model compound in solution, suggesting an SN2 like mechanism for the phosphoryl transfer reaction catalyzed by myosin.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
