Participation of Tryptophan Residues in Dehydrogenase Action

II. POSITION OF TRITIUM-LABELED HYDROGEN IN YEAST ALCOHOL DEHYDROGENASE AFTER VARIOUS MEANS OF INACTIVATION AND HYDROLYSIS

From the ¹ From the Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

A previous study with substrate quantities of yeast alcohol dehydrogenase and tritium-labeled ethanol had shown that the dehydrogenase protein became labeled in the methylene group of a tryptophan residue by the hydrogen atom that is transferred. The labeled protein was precipitated when the enzymatic reaction at equilibrium at pH 9 to 11 was interrupted by heat or addition of HClO₄ (1). The present investigation showed a similar labeling when the enzyme was inactivated by 6 m guanidinium chloride or 0.8 m sodium hydroxide. In all four cases studied, the label appeared in tryptophan after acid hydrolysis of the protein. Inactivation of the enzyme by N-bromosuccinimide or silver nitrate did not result in labeled protein.

Since classical enzymatic and alkaline hydrolytic methods gave unsatisfactory or inconclusive results, a new method of nonacidic protein hydrolysis (2.4 m sodium phosphate, pH 12, 3 to 20 hours at 111°) was developed and applied to the system. The tritium label was also found in the methylene group of tryptophan after heat precipitation and phosphate hydrolysis of the enzyme.

Further study of incomplete enzymatic reactions confirmed the requirement for an equilibrium mixture of substrates and coenzymes for the labeling of tryptophan. Experiments with omission of substrates, with tritium-labeled diphosphopyridine nucleotide as a source of label, showed that perchloric acid precipitated the protein in labeled form. This labeling reaction differed from that of the complete system in that the labeled residue was not tryptophan. Similar experiments with omission of DPN, and with ethanol-1-³H and acetaldehyde, did not yield labeled protein.

Tryptophan uniformly labeled with tritium was converted into N-acetyltryptophan, N-acetyl-5,7-dibromodioxindolylalanine lactone, and indole, and the radioactivity of these derivatives was determined. It was established that indole does not exchange the tritium label with solvent protons during its formation.

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