The pyruvate dehydrogenase from Escherichia coli
showed a primary kinetic isotope effect when its
overall reaction or the partial reaction of the pyruvate
dehydrogenase component were tested in deuterium
oxide. The Michaelis constants for pyruvate were
nearly unchanged, but the maximum velocities in water
and deuterium oxide differed, their ratio being DV =
1.7 for the overall reaction and DV = 2.1 for the E1p reaction.
The pH profile and, accordingly, the δpK1 and
δpK2 values were shifted by 0.6 units to higher pL values.
A linear proton inventory curve was obtained
when varying the atom fractions of protons relative to
deuterons from 100 to 0%. This is an indication for a
single proton transfer. It is proposed that this relatively
weak primary isotope effect may be caused by the
protonation of the N1 nitrogen at the pyrimidine ring
of the cofactor by an adjacent glutamate residue. The
proton of its carboxylic group exchanges very fast
with deuterons of the solvent.
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