Source:http://linkedlifedata.com/resource/pubmed/id/12051945
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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
1
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| pubmed:dateCreated |
2002-6-7
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| pubmed:abstractText |
The active site of glucosamine-6-phosphate deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) has a complex lid formed by two antiparallel beta-strands connected by a helix-loop segment (158-187). This motif contains Arg172, which is a residue involved in binding the substrate in the active-site, and three residues that are part of the allosteric site, Arg158, Lys160 and Thr161. This dual binding role of the motif forming the lid suggests that it plays a key role in the functional coupling between active and allosteric sites. Previous crystallographic work showed that the temperature coefficients of the active-site lid are very large when the enzyme is in its T allosteric state. These coefficients decrease in the R state, thus suggesting that this motif changes its conformational flexibility as a consequence of the allosteric transition. In order to explore the possible connection between the conformational flexibility of the lid and the function of the deaminase, we constructed the site-directed mutant Phe174-Ala. Phe174 is located at the C-end of the lid helix and its side-chain establishes hydrophobic interactions with the remainder of the enzyme. The crystallographic structure of the T state of Phe174-Ala deaminase, determined at 2.02 A resolution, shows no density for the segment 162-181, which is part of the active-site lid (PDB 1JT9). This mutant form of the enzyme is essentially inactive in the absence of the allosteric activator, N-acetylglucosamine-6-P although it recovers its activity up to the wild-type level in the presence of this ligand. Spectrometric and binding studies show that inactivity is due to the inability of the active-site to bind ligands when the allosteric site is empty. These data indicate that the conformational flexibility of the active-site lid critically alters the binding properties of the active site, and that the occupation of the allosteric site restores the lid conformational flexibility to a functional state.
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| pubmed:commentsCorrections | |
| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical | |
| pubmed:status |
MEDLINE
|
| pubmed:month |
May
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| pubmed:issn |
0022-2836
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| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
24
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| pubmed:volume |
319
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
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| pubmed:pagination |
183-9
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| pubmed:dateRevised |
2011-11-17
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| pubmed:meshHeading |
pubmed-meshheading:12051945-Alanine,
pubmed-meshheading:12051945-Aldose-Ketose Isomerases,
pubmed-meshheading:12051945-Allosteric Regulation,
pubmed-meshheading:12051945-Allosteric Site,
pubmed-meshheading:12051945-Circular Dichroism,
pubmed-meshheading:12051945-Crystallography, X-Ray,
pubmed-meshheading:12051945-Enzyme Activation,
pubmed-meshheading:12051945-Escherichia coli,
pubmed-meshheading:12051945-Kinetics,
pubmed-meshheading:12051945-Models, Molecular,
pubmed-meshheading:12051945-Mutagenesis, Site-Directed,
pubmed-meshheading:12051945-Mutation,
pubmed-meshheading:12051945-Phenylalanine,
pubmed-meshheading:12051945-Pliability,
pubmed-meshheading:12051945-Protein Conformation,
pubmed-meshheading:12051945-Structure-Activity Relationship
|
| pubmed:year |
2002
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| pubmed:articleTitle |
On the role of the conformational flexibility of the active-site lid on the allosteric kinetics of glucosamine-6-phosphate deaminase.
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| pubmed:affiliation |
Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, P.O. Box 70-159, Ciudad Universitaria, Mexico City, D.F. 04510, Mexico. ismaelb@servidor.unam.mx
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| pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, U.S. Gov't, Non-P.H.S.,
Research Support, Non-U.S. Gov't
|