2oz0

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

Overview

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.

About this Structure

2OZ0 is a Single protein structure of sequence from Saccharomyces cerevisiae with HEM, FMN and PYR as ligands. Active as L-lactate dehydrogenase (cytochrome), with EC number 1.1.2.3 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 Wed Nov 21 13:22:17 2007