TI: EXTREME PKA DISPLACEMENTS AT THE ACTIVE-SITES OF FMN-DEPENDENT
ALPHA-HYDROXY ACID-OXIDIZING ENZYMES
AU: LEDERER_F
NA: HOP NECKER ENFANTS MALAD,CNRS,URA 1461,161 RUE SEVRES,F-75743
PARIS 15,FRANCE
JN: PROTEIN SCIENCE 1992 Vol.1 No.4 pp.540-548
AB: Flavocytochrome b2 (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
b2, 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 pKa 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 b2. The functional significance of these pK
shifts in terms of catalysis and semiquinone stabilization is
discussed.
KP: SPINACH GLYCOLATE OXIDASE, BAKERS-YEAST, FLAVOCYTOCHROME B2,
ELECTRON-TRANSFER, LACTATE OXIDASE, NUCLEOTIDE-SEQUENCE,
HANSENULA-ANOMALA, PROSTHETIC GROUPS, NMR-SPECTROSCOPY,
TRANSITION-STATE
WA: ALPHA-HYDROXY ACID OXIDATION, FLAVOCYTOCHROME B2, FLAVOENZYMES,
IONIZATION CONSTANTS, PROTON TRANSFER
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RF: 1517_92 2 TORPEDO ACETYLCHOLINESTERASE, CATALYTIC TRIAD,
LIPOPROTEIN-LIPASE GENE, SITE-DIRECTED MUTAGENESIS,
MISSENSE MUTATIONS, FAMILIAL CHYLOMICRONEMIA
4074_92 1 14-KDA GROUP-II PHOSPHOLIPASE-A2, MICELLAR LIPID WATER
INTERFACES OF COMPETITIVE INHIBITORS, IMMUNOCHEMICAL
QUANTITATION