JBC, Vol. 250, Issue 18, 7266-7271, Sep, 1975
Lactate dehydrogenase-catalyzed stereospecific hydrogen atom transfer from reduced nicotinamide adenine dinucleotide to dicarboxylate radicals
P. C. Chan and B. H. Bielski
The dicarboxylate radical -OOC--CH--CH(OH)COO- was generated in an
N2O-saturated fumarate solution by high energy ionizing radiation. When
NADH was present in the solution, product analysis indicated a
stoichiometry of 2 molecules of the radical reacted with 1 NADH molecule to
form 2 malate and 1 enzymatically active NAD+ molecules. In a similar
experiment using tritium label on position A of NADH, due to an isotope
effect, only 10% of the label was transferred to malate; most of the
remaining tritium was found in the NAD+ formed. When lactate dehydrogenase
was added, however, no la bel was detectable in NAD+, and over 80% of the
tritium lost from NADH was found in malate. The stereospecific transfer of
the hydrogen atom from lactate dehydrogenase-bound NADH to the
dicarboxylate radical suggested that the free radical reaction must have
taken place at the active site. The hydrogen atom transfer was inhibited by
oxamate. Results from flow experiments in which an irradiated fumarate
solution was mixed with a solutionof lactate dehydrogenase and NADH are in
support of a mechanism in which the hydrogen atom transfer occurs in the
first oxidation step.
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