Bovine inositol monophosphatase : structural and functional studies.
Bovine inositol monophosphatase : structural and functional studies.
Inositol monophosphatase, a homodimeric enzyme of subunit M, 30kDa which catalyses the final dephosphorylation of Ins(1)P and Ins(4)P in the phosphatidylinositol cells signalling pathway, requires at least Mg2+ ions per monomer for catalytic activity. The first binding site for these ions is characterised by a Kd=300μM, as exemplified by fluorescence studies using pyrene maleimide-labelled enzyme and near-UV CD spectroscopy. Metal ion interactions with site 2 (Kd=3mM) may be monitored by kinetic analyses using 4-methylumbelliferyl phosphate as the substrate, and by far-UV CD spectroscopy. H217Q and W219F-substituted enzymes have been used to confirm the importance of two metal binding sites, since metal binding parameters associated only with site 2 are affected by these amino acid replacements.
Stopped-flow studies using pyrene-labelled enzyme have been used to show that the enhancement of fluorescence at 380nm on addition of Mg2+ ions occurs in two identifiable phases. The first phase occurs at a rate dependent upon the concentration of these ions, whereas the rate of the second phase is independent of Mg2+ concentration, suggesting a conformational change elicited on the binding of Mg2+ at site 1. Li+ ions, which inhibit the enzyme uncompetitively at concentrations below 5mM with respect to Ins(1)P, have been shown by pyrene fluorescence studies to only have a low affinity for site 1 (Kd=100mM), whereas the IC50 value for these ions with WT-enzyme is 16mM when 4-methylumbelliferyl phosphate is used as substrate, suggesting that these ions inhibit the enzyme by binding at site 2. Limited proteolysis studies using trypsin have shown that the loss of the N-terminal 36 residues leads to the destruction of both metal sites 1 and 2, thereby resulting in the inactivation of the enzyme.
University of Southampton
1996
Thorne, Mark Richard
(1996)
Bovine inositol monophosphatase : structural and functional studies.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Inositol monophosphatase, a homodimeric enzyme of subunit M, 30kDa which catalyses the final dephosphorylation of Ins(1)P and Ins(4)P in the phosphatidylinositol cells signalling pathway, requires at least Mg2+ ions per monomer for catalytic activity. The first binding site for these ions is characterised by a Kd=300μM, as exemplified by fluorescence studies using pyrene maleimide-labelled enzyme and near-UV CD spectroscopy. Metal ion interactions with site 2 (Kd=3mM) may be monitored by kinetic analyses using 4-methylumbelliferyl phosphate as the substrate, and by far-UV CD spectroscopy. H217Q and W219F-substituted enzymes have been used to confirm the importance of two metal binding sites, since metal binding parameters associated only with site 2 are affected by these amino acid replacements.
Stopped-flow studies using pyrene-labelled enzyme have been used to show that the enhancement of fluorescence at 380nm on addition of Mg2+ ions occurs in two identifiable phases. The first phase occurs at a rate dependent upon the concentration of these ions, whereas the rate of the second phase is independent of Mg2+ concentration, suggesting a conformational change elicited on the binding of Mg2+ at site 1. Li+ ions, which inhibit the enzyme uncompetitively at concentrations below 5mM with respect to Ins(1)P, have been shown by pyrene fluorescence studies to only have a low affinity for site 1 (Kd=100mM), whereas the IC50 value for these ions with WT-enzyme is 16mM when 4-methylumbelliferyl phosphate is used as substrate, suggesting that these ions inhibit the enzyme by binding at site 2. Limited proteolysis studies using trypsin have shown that the loss of the N-terminal 36 residues leads to the destruction of both metal sites 1 and 2, thereby resulting in the inactivation of the enzyme.
This record has no associated files available for download.
More information
Published date: 1996
Identifiers
Local EPrints ID: 460148
URI: http://eprints.soton.ac.uk/id/eprint/460148
PURE UUID: 8b29ccc4-2bd8-4005-8c7f-c7c9f2c62d38
Catalogue record
Date deposited: 04 Jul 2022 18:01
Last modified: 04 Jul 2022 18:01
Export record
Contributors
Author:
Mark Richard Thorne
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics