Biosynthetic labeling of diphthamide in Saccharomyces cerevisiae.
Diphthamide, the post-translational amino acid derivative in the diphtheria toxin-modification site of protein synthesis elongation factor 2 (EF-2), has the proposed structure 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, 10710-10716). The identification of the biosynthetic precursors of diphthamide would provide a means of evaluating its proposed structure and determining if the amino acid occurs in proteins other than EF-2. Toward this end, yeast were grown on potential radiolabeled precursors and the resulting radiolabeled protein was hydrolyzed in acid. The acid hydrolysates were subjected to amino acid analysis with a program optimized to resolve diphthine, the acid hydrolysis product of diphthamide, from the common amino acids. Radiolabel from [beta-3H]histidine, [alpha-3H]methionine, and [Me-3H] methionine was found to be incorporated into diphthine in a molar ratio of 1:1:3 while that of [35S]methionine was not incorporated. These results are in accord with the proposed structure of diphthamide and suggest that in its biosynthesis the backbone and 3 methyl groups of methionine are added to a histidine residue in the peptide chain of EF-2. These labeling experiments show that diphthine (diphthamide) constitutes approximately 6 X 10(-6) mol fraction of the total amino acids in yeast protein hydrolysates. Estimates of the amount of diphthamide present in the diphtheria toxin-modification site of EF-2 indicate that it constitutes from 4.5 to 9 X 10(-6) mol fraction of the total amino acids in yeast protein. The present evidence suggests that diphthamide occurs only in EF-2.
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D006639:Histidine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D006868:Hydrolysis; D008715:Methionine / Q000378:metabolism; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D000596:Amino Acids; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D006639:Histidine; C027527:diphthamide; D008715:Methionine