Linked Life Data

14581486

Source:http://linkedlifedata.com/resource/pubmed/id/14581486

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2004-1-5
pubmed:abstractText
A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitivity in solution. Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy. Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C. The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering. Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is activated by ATP and inhibited by CTP at subsaturating concentrations of aspartate. The binding of the bisubstrate analogue N-(phosphonoacetyl)-l-aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrenelabeled enzyme caused a sigmoidal change in the fluorescence emission. Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate and carbamoyl phosphate) caused a hyperbolic increase and decrease, respectively, in the fluorescence emission. The half-saturation values from the fluorescence saturation curves and kinetic saturation curves were, within error, identical. Fluorescence and small-angle x-ray scattering stopped-flow experiments, using aspartate and carbamoyl phosphate, confirm that the change in excimer fluorescence and the quaternary structure change correlate. These results in conjunction with previous studies suggest that the allosteric transition involves both global and local conformational changes and that the heterotropic effect of the nucleotides may be exerted through local conformational changes in the active site by directly influencing the conformation of the 240s loop.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
9
pubmed:volume
279
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
945-51
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:14581486-Adenosine Triphosphate, pubmed-meshheading:14581486-Allosteric Site, pubmed-meshheading:14581486-Aspartate Carbamoyltransferase, pubmed-meshheading:14581486-Binding Sites, pubmed-meshheading:14581486-Catalysis, pubmed-meshheading:14581486-Cytidine Triphosphate, pubmed-meshheading:14581486-Dose-Response Relationship, Drug, pubmed-meshheading:14581486-Escherichia coli, pubmed-meshheading:14581486-Fluorescent Dyes, pubmed-meshheading:14581486-Kinetics, pubmed-meshheading:14581486-Models, Molecular, pubmed-meshheading:14581486-Mutation, pubmed-meshheading:14581486-Plasmids, pubmed-meshheading:14581486-Protein Binding, pubmed-meshheading:14581486-Protein Conformation, pubmed-meshheading:14581486-Pyrenes, pubmed-meshheading:14581486-Scattering, Radiation, pubmed-meshheading:14581486-Spectrometry, Fluorescence, pubmed-meshheading:14581486-Time Factors, pubmed-meshheading:14581486-X-Rays
pubmed:year
2004
pubmed:articleTitle
A fluorescent probe-labeled Escherichia coli aspartate transcarbamoylase that monitors the allosteric conformational state.
pubmed:affiliation
Department of Chemistry, Boston College, Merkert Chemistry Center, Chestnut Hill, Massachusetts 02467, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.