The molecular properties of NAD-coenzymes bound to various dehydrogenases: A classical Raman difference study.
Item
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Title
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The molecular properties of NAD-coenzymes bound to various dehydrogenases: A classical Raman difference study.
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Identifier
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AAI9119695
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identifier
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9119695
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Creator
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Zheng, Jie.
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Contributor
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Adviser: Robert H. Callender
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Date
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1991
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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 | Physics, Molecular | Chemistry, Biochemistry
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Abstract
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The Raman spectrum of a small molecule or molecular moiety when bound to proteins can be measured by using difference techniques developed in our laboratory. In the thesis, we have applied this method in obtaining the Raman spectra of NAD(P)-coenzymes and their analogies when bound to lactate dehydrogenase (LDH) and dihydrofolate reductase (DHFR). The binding of the coenzymes to the proteins causes significant changes in the Raman spectra of these molecules relative to spectra obtained in the absence of enzymes. When the coenzymes bind to LDH, the molecular motions of the adenine moiety of both NAD+ and NADH as well as adenine containing analogues of these coenzymes produce Raman bands that are essentially identical to each other but very different from solution spectra, suggesting that the binding of adenine to the enzyme is the same regardless of the nicotinamide head-group nature. Protonation of the bound adenine ring at the 3-position is offered as a possible explanation for the changes observed in the Raman spectra of adenine when it binds to these two enzymes relative to its solution spectrum. Significant shifts are observed in both the stretching frequency of the carboxamide carbonyl of the NAD+ and analogues and the rocking motion of the carboxamide NH{dollar}\sb2{dollar} group of NADH. We ascribe these shifts to the formation of strong hydrogen bonding between the cofactor's carboxamide group and the apoenzyme. We suggest that this hydrogen bonding is responsible for the very high degree of stereochemistry fidelity observed with this enzyme. When the coenzymes bound to DHFR, the binding properties of nicotinamide-head are found to be quite similar to those bound for LDH. However, the adenine moiety shows different Raman spectra. We suggest that when the cofactor binds to the DHFR, the adenine moiety fits into a hydrophobic pocket and does not change its protanation state. Finally, we use the C-H (C-D) stretching mode at the C4 position of nicotinamide to probe the possibility of the pucker state of the nicotinamide ring when it binds to dehydrogenases. We find considerable ring puckers for NADH upon binding to LDH and DHFR and little ring puckering for NADH in solution. We discuss this in relationship to the enzymatic properties of LDH and DHFR.
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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.
