Tryptophan as intermediate in dehydrogenase action. 3. Evidence for complete cycle of hydrogen transfer between substrate and tryptophanyl residues in rabbit muscle lactate dehydrogenase.

Rabbit muscle lactate dehydrogenase, following equilibration at pH 10 with lactate-2-3H, was precipitated by perchloric acid or reversibly denatured by 8 M urea. The enzyme protein after either method of inactivation was found to contain about 0.5 g atom of tritium label per 132,000 g of protein. The urea-denatured, labeled enzyme, after isolation by Sephadex chromatography and partial reactivation by dilution, was shown by “all-or-none” assay to transfer 63 % of its tritium back to lactate or reduced nicotinamide adenine dinucleotide. After acid or alkaline phosphate hydrolysis of the perchloric acidor urea-denatured enzyme, 12 to 32% of the label was found in tryptophan isolated from the hydrolysate. Tritium assay of N-acetyl-DL-tryptophan and indole prepared from a sample of the labeled tryptophan indicated that the tritium was in the methylene group of the tryptophan molecule. These experiments suggest the participation of a tryptophanyl residue of muscle lactate dehydrogenase during reversible hydrogen transfer between lactate and NAD, via oxidation to an indolenine. This result is similar to that previously found for yeast alcohol dehydrogenase (2, 3).