CHEMICAL AND KINETIC STUDIES OF BACTERIAL TRANSPORT AND CHEMORECEPTOR PROTEINS
type: CreativeWork
description: The L-arabinose-binding protein, an essential component of the high affinity L-arabinose transport system of Escherichia coli, forms a covalent adduct with the chromophoric probe 2-chloromercuri-4-nitrophenol (MNP) at a single site, cysteine residue 64. The addition of L-arabinose to MNP-modified L-arabinose-binding protein causes an increase in the nitrophenol pK to approximately 8.0 and a blue shift in the wavelength of maximum absorbance of the nitrophenolate species. The rate of reaction of MNP or 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) with native L-arabinose-binding protein in the presence L-arabinose was found to be inversely related to the concentration of sugar. In addition, L-arabinose-binding activity is dramatically reduced by thiol reagents; an SCN derivative, prepared by reaction of KCN with thionitrobenzoate-modified binding protein was less inhibited than binding protein modified with either MNP or DTNB. The residual binding activity and the apparent dependence upon the size of the group attached to the cysteine suggests that whereas the cysteine is not "essential" it is near the binding site. This conclusion has been confirmed by X-ray crystallographic data. The reactions of specific ligands with the L-arabinose-, D-galactose-,D-maltose- and L-histidine-binding proteins were examined by stopped flow rapid mixing techniques. The change in tryptophan fluorescence that occurs in these reactions is adequately described in all cases by a rapid second order process, no additional phases of fluorescence charge were observed. The resultant time courses were fitted to a simple equilibrium model of binding with association rate constants on the order of 10('7) to 10('8) M('-1)s('-1) and dissociation rate constants in the range of 1 to 100 s('-1). These findings indicate that the ligand-induced conformational changes proposed for these proteins must proceed at a rapid rate. It was also established that purified L-arabinose('-), D-galactose('-), and L-histi…
abstract: The L-arabinose-binding protein, an essential component of the high affinity L-arabinose transport system of Escherichia coli, forms a covalent adduct with the chromophoric probe 2-chloromercuri-4-nitrophenol (MNP) at a single site, cysteine residue 64. The addition of L-arabinose to MNP-modified L-arabinose-binding protein causes an increase in the nitrophenol pK to approximately 8.0 and a blue shift in the wavelength of maximum absorbance of the nitrophenolate species. The rate of reaction of MNP or 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) with native L-arabinose-binding protein in the presence L-arabinose was found to be inversely related to the concentration of sugar. In addition, L-arabinose-binding activity is dramatically reduced by thiol reagents; an SCN derivative, prepared by reaction of KCN with thionitrobenzoate-modified binding protein was less inhibited than binding protein modified with either MNP or DTNB. The residual binding activity and the apparent dependence upon the size of the group attached to the cysteine suggests that whereas the cysteine is not "essential" it is near the binding site. This conclusion has been confirmed by X-ray crystallographic data. The reactions of specific ligands with the L-arabinose-, D-galactose-,D-maltose- and L-histidine-binding proteins were examined by stopped flow rapid mixing techniques. The change in tryptophan fluorescence that occurs in these reactions is adequately described in all cases by a rapid second order process, no additional phases of fluorescence charge were observed. The resultant time courses were fitted to a simple equilibrium model of binding with association rate constants on the order of 10('7) to 10('8) M('-1)s('-1) and dissociation rate constants in the range of 1 to 100 s('-1). These findings indicate that the ligand-induced conformational changes proposed for these proteins must proceed at a rapid rate. It was also established that purified L-arabinose('-), D-galactose('-), and L-histi…
author: MILLER, DAVID MILTON, III
language: eng
keywords: Biochemistry
date published: 1981
date modified: 2026-07-24T04:10:17Z
provider: Rice University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Rice University

PropertyValue
property ID: Handle
value: https://hdl.handle.net/1911/15634