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| - | [[Image:2oz0.jpg|left|200px]] | + | {{Seed}} |
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| | {{STRUCTURE_2oz0| PDB=2oz0 | SCENE= }} | | {{STRUCTURE_2oz0| PDB=2oz0 | SCENE= }} |
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| - | '''Mechanistic and Structural Studies of H373Q Flavocytochrome b2: Effects of Mutating the Active Site Base'''
| + | ===Mechanistic and Structural Studies of H373Q Flavocytochrome b2: Effects of Mutating the Active Site Base=== |
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| - | ==Overview==
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| - | His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton. The effects of mutating this residue to glutamine have been determined to provide further insight into its role. The kcat and kcat/Klactate values for the mutant protein are 3 to 4 orders of magnitude smaller than the wild-type values, consistent with a critical role for His373. Similar effects are seen when the mutation is incorporated into the isolated flavin domain of the enzyme, narrowing the effects to lactate oxidation rather than subsequent electron transfers. The decrease of 3500-fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most effected by the mutation. The primary deuterium and solvent kinetic isotope effects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q flavocytochrome b2 than in the wild-type enzyme. The structure of the mutant enzyme with pyruvate bound, determined at 2.8 A, provides a rationale for these effects. The orientation of pyruvate in the active site is altered from that seen in the wild-type enzyme. In addition, the active site residues Arg289, Asp 292, and Leu 286 have altered positions in the mutant protein. The combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_17563122}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 17563122 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_17563122}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Oxidoreductase]] | | [[Category: Oxidoreductase]] |
| | [[Category: Pyruvate]] | | [[Category: Pyruvate]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 11:57:56 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 07:58:54 2008'' |
Revision as of 04:58, 29 July 2008
Template:STRUCTURE 2oz0
Mechanistic and Structural Studies of H373Q Flavocytochrome b2: Effects of Mutating the Active Site Base
Template:ABSTRACT PUBMED 17563122
About this Structure
2OZ0 is a Single protein structure of sequence from Saccharomyces cerevisiae. Full crystallographic information is available from OCA.
Reference
Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base., Tsai CL, Gokulan K, Sobrado P, Sacchettini JC, Fitzpatrick PF, Biochemistry. 2007 Jul 3;46(26):7844-51. Epub 2007 Jun 12. PMID:17563122
Page seeded by OCA on Tue Jul 29 07:58:54 2008
