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| - | [[Image:1e0t.gif|left|200px]] | + | {{Seed}} |
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| | {{STRUCTURE_1e0t| PDB=1e0t | SCENE= }} | | {{STRUCTURE_1e0t| PDB=1e0t | SCENE= }} |
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| - | '''R292D MUTANT OF E. COLI PYRUVATE KINASE'''
| + | ===R292D MUTANT OF E. COLI PYRUVATE KINASE=== |
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| - | ==Overview==
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| - | Pyruvate kinase (PK) is critical for the regulation of the glycolytic pathway. The regulatory properties of Escherichia coli were investigated by mutating six charged residues involved in interdomain salt bridges (Arg(271), Arg(292), Asp(297), and Lys(413)) and in the binding of the allosteric activator (Lys(382) and Arg(431)). Arg(271) and Lys(413) are located at the interface between A and C domains within one subunit. The R271L and K413Q mutant enzymes exhibit altered kinetic properties. In K413Q, there is partial enzyme activation, whereas R271L is characterized by a bias toward the T-state in the allosteric equilibrium. In the T-state, Arg(292) and Asp(297) form an intersubunit salt bridge. The mutants R292D and D297R are totally inactive. The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure. However, the mutation induces a reorganization of the interface with the creation of a network of interactions similar to that observed in the crystal structures of R-state yeast and M1 PK proteins. Furthermore, in the R292D structure, two loops that are part of the active site are disordered. The K382Q and R431E mutations were designed to probe the binding site for fructose 1, 6-bisphosphate, the allosteric activator. R431E exhibits only slight changes in the regulatory properties. Conversely, K382Q displays a highly altered responsiveness to the activator, suggesting that Lys(382) is involved in both activator binding and allosteric transition mechanism. Taken together, these results support the notion that domain interfaces are critical for the allosteric transition. They couple changes in the tertiary and quaternary structures to alterations in the geometry of the fructose 1, 6-bisphosphate and substrate binding sites. These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_10751408}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 10751408 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_10751408}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Allostery]] | | [[Category: Allostery]] |
| | [[Category: Glycolysis]] | | [[Category: Glycolysis]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 14:31:12 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jun 30 23:57:22 2008'' |
Revision as of 20:57, 30 June 2008
Template:STRUCTURE 1e0t
R292D MUTANT OF E. COLI PYRUVATE KINASE
Template:ABSTRACT PUBMED 10751408
About this Structure
1E0T is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
Reference
The allosteric regulation of pyruvate kinase., Valentini G, Chiarelli L, Fortin R, Speranza ML, Galizzi A, Mattevi A, J Biol Chem. 2000 Jun 16;275(24):18145-52. PMID:10751408
Page seeded by OCA on Mon Jun 30 23:57:22 2008
