RAMAN SPECTROSCOPIC STUDIES OF EQUINE LIVER ALCOHOL DEHYDROGENASE, ITS COENZYMES AND SUBSTRATES.
Item
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Title
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RAMAN SPECTROSCOPIC STUDIES OF EQUINE LIVER ALCOHOL DEHYDROGENASE, ITS COENZYMES AND SUBSTRATES.
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Identifier
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AAI8801736
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identifier
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8801736
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Creator
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MARTIN, CHARLOTTE LEVIAN.
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Contributor
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Robert H. Callender | Donald L. Sloan
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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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Chemistry, Biochemistry
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Abstract
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The solution Raman spectrum of equine liver alcohol dehydrogenase (EC-1.1.1.1., LADH) has been obtained. Its secondary structure as determined by a close examination of the Raman bands is somewhat different from its crystalline structure, as determined by X-ray diffraction.;The binary complex of the coenzyme, reduced nicotinamide adenine dinucleotide (NADH) and LADH has been studied. Using difference Raman spectroscopy, the Raman features of NADH bound to LADH have been determined. There are significant changes in the bound NADH spectrum relative to its solution spectrum. The data suggests that both the nicotinamide and the adenine moieties of NADH are involved in binding and at least one of the two NH{dollar}\sb2{dollar} moieties is involved as well.;In order to understand the spectroscopy and to fully interpret the data in molecular terms as they relate to the organic mechanism occurring at the active site of the enzyme, LADH, Raman spectroscopy of reduced nicotinamide adenine dinucleotide, NADH, and oxidized nicotinamide adenine dinucleotide, NAD{dollar}\sp+{dollar}, has been systematically carried out through the study of various fragments and analogues of these molecules. The effects of deuteration and pH on the exchangeable protons of NADH and NAD{dollar}\sp+{dollar} also have been studied. By comparing the relative intensities and positions of the peaks in the Raman spectra of NADH and NAD{dollar}\sp+{dollar} with those of their fragments and analogues, it has been found useful to consider these rather complicated molecules (NADH and NAD{dollar}\sp+{dollar}) as consisting primarily of the various components: adenine, two riboses, two phosphates and nicotinamide for the purpose of assigning their Raman bands.;The aromatic aldehyde p-(dimethylamino) benzaldehyde (DABA) a relatively poor substrate of LADH has been studied in an attempt at understanding the molecular details of this bound substrate and how it and other substrates bind at the active site of LADH. This compound when complexed to Zn{dollar}\sp{lcub}2+{rcub}{dollar} in diethyl ether has been used previously as a model for the enzyme substrate complex between LADH and DABA (46,47). Therefore, as a model of the substrate in the molecular environment of the enzyme active site the prereasonance Raman spectra of the native DABA when ligated with zinc ion in methylene chloride and diethyl ether has been obtained. In addition, various isotopically labelled compounds of DABA ({dollar}\sp{13}{dollar}C, {dollar}\sp2{dollar}H) have been studied in order to interpret the Raman spectra of DABA and the DABA-Zn{dollar}\sp{lcub}++{rcub}{dollar} models. We find that the DABA-Zn{dollar}\sp{lcub}++{rcub}{dollar} complexes in solution are excellent models for events which may occur in situ.
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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.
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Program
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Biochemistry
