Lactate dehydrogenase-catalyzed stereospecific hydrogen atom transfer from reduced nicotinamide adenine dinucleotide to dicarboxylate radicals.

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.