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| | {{STRUCTURE_1l8p| PDB=1l8p | SCENE= }} | | {{STRUCTURE_1l8p| PDB=1l8p | SCENE= }} |
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| - | '''Mg-phosphonoacetohydroxamate complex of S39A yeast enolase 1'''
| + | ===Mg-phosphonoacetohydroxamate complex of S39A yeast enolase 1=== |
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| - | ==Overview==
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| - | Crystallographic and kinetic methods have been used to characterize a site-specific variant of yeast enolase in which Ser 39 in the active-site flap has been changed to Ala. In the wild-type enzyme, the carbonyl and hydroxyl groups of Ser 39 chelate the second equivalent of divalent metal ion, effectively anchoring the flap over the fully liganded active site. With Mg(2+) as the activating cation, S39A enolase has <0.01% of wild-type activity as reported previously [J.M. Brewer, C.V. Glover, M.J. Holland, L. Lebioda, Biochim. Biophys. Acta 1383 (2) (1998) 351-355]. Measurements of (2)H kinetic isotope effects indicate that the proton abstraction from 2-phosphoglycerate (2-PGA) is significantly rate determining. Analysis of the isotope effects provides information on the relative rates of formation and breakdown of the enolate intermediate. Moreover, assays with different species of divalent metal ions reveal that with S39A enolase (unlike the case of wild-type enolase), more electrophilic metal ions promote higher activities. The kinetic results with the S39A variant support the notions that a rate-limiting product release lowers the activity of wild-type enolase with more electrophilic metal ions and that the metal ions are used to acidify the C2-proton of 2-PGA. The S39A enolase was co-crystallized with Mg(2+) and the inhibitor phosphonoacetohydroxamate (PhAH). The structure was solved and refined at a resolution of 2.1 A. The structure confirms the conjecture that the active-site flap is opened in the mutant protein. PhAH chelates to both Mg ions as in the corresponding structure of the wild-type complex. Positions of the side chains of catalytic groups, Lys 345 and Glu 211, and of "auxiliary" residues Glu 168 and Lys 396 are virtually unchanged relative to the complex with the wild-type protein. His 159, which hydrogen bonds to the phosphonate oxygens in the wild-type complex, is 5.7 A from the closest phosphonate oxygen, and the loop (154-166) containing His 159 is shifted away from the active center. A peripheral loop, Glu 251-Gly 275, also moves to open access to the active site.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_12054465}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 12054465 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_12054465}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Wong, S W.]] | | [[Category: Wong, S W.]] |
| | [[Category: Beta barrel]] | | [[Category: Beta barrel]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 23:40:20 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Wed Jul 2 11:57:32 2008'' |
Revision as of 08:57, 2 July 2008
Template:STRUCTURE 1l8p
Mg-phosphonoacetohydroxamate complex of S39A yeast enolase 1
Template:ABSTRACT PUBMED 12054465
About this Structure
1L8P is a Single protein structure of sequence from Saccharomyces cerevisiae. Full crystallographic information is available from OCA.
Reference
Functional and structural changes due to a serine to alanine mutation in the active-site flap of enolase., Poyner RR, Larsen TM, Wong SW, Reed GH, Arch Biochem Biophys. 2002 May 15;401(2):155-63. PMID:12054465
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