Protein Science, Vol 1, Issue 4 540-548, Copyright © 1992 by Cold Spring Harbor Laboratory Press

ARTICLE

Extreme pK(a) displacements at the active sites of FMN-dependent {alpha}-hydroxy acid-oxidizing enzymes

F. LEDERER
CNRS URA 1461, Hopital Necker, 161 Rue de Sevres, 75743 Paris Cedex 15, France

Flavocytochrome b(2) (or L-lactate dehydrogenase) from baker's yeast is thought to operate by the initial formation of a carbanion, as do the evolutionarily related {alpha}-hydroxy acid-oxidizing FMN-dependent oxidases. Previous work has shown that, in the active site of the unligated reduced flavocytochrome b(2), the group that has captured the substrate {alpha}-proton has a high pK(app), calculated to lie around 15 through the use of Eigen's equation. A detailed inspection of the now known three-dimensional structure of the enzyme leads to the conclusion that the high pK(a) belongs to His 373, an active site group that plays the role of general base in the forward reaction and of general acid in the reverse direction. Moreover, consideration of the kinetics of proton transfer during the catalytic cycle suggests that the pK(a) of the reduced FMN N5 position should be lowered by several pH units compared to its pK(a) of 20 or more when free. The features of the three-dimensional structure possibly responsible for these pK shifts are analyzed; they are proposed to consist of a network of hydrogen bonds with the solvent and of a mutual electrostatic stabilization of anionic reduced flavin and the imidazolium ion. Finally, it is suggested that similar pK shifts affect the active sites of the {alpha}-hydroxy acid-oxidizing flavooxidases, which are homologous to flavocytochrome b(2). The functional significance of these pK shifts in terms of catalysis and semiquinone stabilization is discussed.
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