Studies of substrates bound to horse liver alcohol dehydrogenase: Raman spectroscopy of NAD(+), NADH and DABA in situ.
Item
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Title
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Studies of substrates bound to horse liver alcohol dehydrogenase: Raman spectroscopy of NAD(+), NADH and DABA in situ.
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Identifier
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AAI8801690
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identifier
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8801690
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Creator
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Chen, De Huai.
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Contributor
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Adviser: Robert H. Callender
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Date
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1987
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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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In this thesis research we have studied the Raman spectra of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD{dollar}\sp+{dollar}), adenosine 5{dollar}\sp\prime{dollar}-diphosphate ribose (ADPR) and 1, 4, 5, 6 tetrahydronicotinamide adenine dinucleotide (H{dollar}\sb2{dollar}NADH) when bound to the coenzyme site of liver alcohol dehydrogenase (LADH). The bound NADH spectrum is calculated by comparisons the classical Raman spectrum of the binary complex, LADH/NADH, to that of LADH and by subtracting them. The spectrum of bound ADPR allows an assignment of the bands of the bound NADH and NAD{dollar}\sp+{dollar} spectra to normal coordinates located primarily on either the nicotinamide and adenine moieties. This assignment has been confirmed from the studies of H{dollar}\sb2{dollar}NADH in solution and in situ. By comparing the spectra of the bound coenzyme with model compound data and through the use of deuterated compounds, we confirm and characterize how the adenine moiety is involved in coenzyme binding and discuss the validity of the suggestion that the adenine ring is protonated upon binding. The nicotinamide moiety of NADH shows significant changes upon binding, as evidenced by its spectrum. We find that the aromatic nature of the NAD{dollar}\sp+{dollar} nicotinamide ring is disrupted in the ternary complex, LADH/NAD{dollar}\sp+{dollar}/pyrazole. We discuss various models which are consistent with the data and with the enzymatic mechanism of LADH. We note that the rather dramatic changes in the coenzyme molecular structure, that occur when NADH or NAD{dollar}\sp+{dollar} binds, are not necessarily repeated at other dehydrogenase binding sites. For example, our preliminary results concerning coenzymes binding to yeast alcohol dehydrogenase show much fewer differences between solution and bound coenzymes.;To understand whether the substrate is directly bound to the active Zn(II) metal cation in the enzymatic hydrogenation by LADH, we have studied the pre-resonance Raman spectra of the aromatic aldehyde p-(dimethylamino) benzaldehyde (DABA) and its various isotopically labelled compounds in buffer solution, when bound to LADH/NADH and when complexed to Zn{dollar}\sp{lcub}+2{rcub}{dollar} in methylene chloride. The results suggest that the DABA-Zn complexes, are good models for DABA in situ. We find that DABA's bond character when bound to LADH/NADH retains a significant amount of essential double bond order. (Abstract shortened with permission of author.).
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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.
