The hydrolysis of phosphates in *Ras bound nucleotides and model systems: An FTIR study.
Item
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Title
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The hydrolysis of phosphates in *Ras bound nucleotides and model systems: An FTIR study.
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Identifier
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AAI3024772
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identifier
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3024772
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Creator
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Cheng, Hu.
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Contributor
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Adviser: Robert H. Callender
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Date
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2001
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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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Biophysics, General
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Abstract
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The hydrolysis of dianionic phosphate monoesters is studied using FTIR and Raman spectroscopy. We have measured the frequencies of both symmetric and antisymmetric vibrational modes for the phosphate in various monoesters. The bond length of the bridging P--O bond is computed using an empirical formula. The relationship between bond length and the pKa of the leaving group is investigated. We also measured the symmetric and antisymmetric frequencies of the phosphate modes in PNPP and PP in various DMSO/water mixtures. The hydrolysis rate is measured in parallel to explore the correlation with the bond length.;We have developed the isotope edited IR difference spectroscopy to measure the antisymmetric vibrational modes of phosphates in GTP and GDP bound to Ras. Structural information is obtained from their vibrational frequencies. It turns out that when GTP•Mg and GDP•Mg move from solution into the active site of Ras, the non-bridging O•• P••O angle of the gamma-phosphate of GTP is opened by 2.7°; yet, the angular freedom of the gamma-phosphate is comparable to that in solution. In contrast, the motion of the gamma-phosphate of GTP is highly restricted, suggesting that it positions the gamma-phosphate for nucleophilic attack. The beta,gamma-bridging O--P bond of bound GTP is weakened, being lengthened by 0.005 A in the active site, corresponding to a bond order decrease of 0.012 valence units (v.u.). With the study of phosphate monoesters, the observed binding change is consistent with a Ras-mediated hydrolysis mechanism that parallels that for solution hydrolysis.;The pKa of gamma-phosphate is predicted to be about 2.9 if it acts as a general base in the hydrolysis of GTP in Ras. Our work investigated this mechanism by studying the pH dependence of Ras•GTP•Mg and Ras•GDP•Mg from their vibrational spectra. The results show that the gamma-phosphate is not protonated at pH > 3.3. GTP remains bound to Ras at pH as low as 2.0. A titration study of the Amide I band shows that Ras•GTP•Mg and Ras•GMP•Mg undergoes conformation changes that appear to be two-state reversible transition in the pH range of 5.4--2.6 and a non-reversible transition in the pH range of 2.6--1.7.
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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.
