2oz0 | pdb_00002oz0

Mechanistic and Structural Studies of H373Q Flavocytochrome b2: Effects of Mutating the Active Site Base

spinqualitylabelsall models 2oz0, resolution 2.80Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 2oz0 is a 2 chain structure with sequence from Saccharomyces cerevisiae. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.8Å Ligands: FMN, HEM, PYR Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function CYB2_YEAST Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed 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. 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 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Lactate dehydrogenase 3D structures

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