Inactivation of flavocytochrome b2 with fluoropyruvate. Reaction at the active-site histidine.
Fluoropyruvate inactivated oxidized flavocytochrome b2 (baker's yeast L-lactate dehydrogenase) in a biphasic process yielding convex semilog plots of residual activity versus time. At each reagent concentration, rate constants k1 and k2 for the two phases could be calculated by simulation studies using one of the schemes proposed by Ray and Koshland [J. Biol. Chem. (1961) 236, 1973-1979]: E----E1 (fully active)----E2 (inactive). When plotted as a function of reagent concentration, the values of k2, but not those of k1, showed a saturation effect. Inactivation was slowed down by D-lactate, a competitive inhibitor, and completely prevented by enzyme reduction. While no enzyme chemical modification could be demonstrated for the first step, the inactivation event of the second step could be ascribed to alkylation of a histidine belonging to proteolytic fragment beta of the enzyme. The only histidine present in the fragment sequence is His-373. In the enzyme three-dimensional structure [Xia et al. (1987) Proc. Natl Acad. Sci. USA 84, 2629-2633] His-373 is well located, close to the cofactor, to play the role of the active-site base required by the chemical mechanism. Alternative chemical interpretations of the kinetic scheme are discussed, so is the difference between flavocytochrome b2 inactivation by fluoropyruvate and bromopyruvate.
Urban
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P
Centre National de la Recherche Scientifique Laboratoire Associé 122, Paris, France.
Lederer
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F
D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005415:Flavins / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D007773:Lactates / Q000494:pharmacology; D011485:Protein Binding; D011489:Protein Denaturation; D011773:Pyruvates / Q000494:pharmacology*
D000596:Amino Acids; D004791:Enzyme Inhibitors; D005415:Flavins; D007773:Lactates; D011773:Pyruvates; D006639:Histidine; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)