Elongation factor 2 (EF-2), the protein which promotes the translocation step during protein synthesis in the cytoplasm of eukaryotic cells, contains an unusual amino acid, designated X by Robinson, Hendriksen, and Maxwell (J. Biol. Chem. 249, 5088, 1974). Amino acid X appears to be essential to the function of EF-2 because its covalent modification by diphtheria toxin inactivates the protein. Although the toxin will apparently inactivate all eukaryotic EF-2s, through this modification, it does not appear to modify any other protein. This suggests that all EF-2s contain a functionally essential and unique structural feature which is specifically recognized by diphtheria toxin. The toxin inactivates EF-2 by transferring the -ADP-ribose of NAD+ to the unusual amino acid X. Amino acid X almost certainly arises by posttranslational modification of a precursor of EF-2 because it is not one of the 20 common amino acids. Interestingly, it also appears to differ from the previously recognized amino acids known to arise by posttranslational modification of proteins. We have developed a large scale method for the purification of amino acid X from yeast EF-2. The initial goal of this proposal is to utilize the material we have prepared by this method to determine the structure of amino acid X and the mode of ADP ribose attachment. For this purpose we will employ primarily nuclear magnetic resonance spectroscopy and mass spectroscopy. Immediately following the determination of the structure of amino acid X we propose to initiate studies designed to determine: 1) The distribution of amino acid X in other proteins of various biological materials; 2) The mechnism of biosynthesis of amino acid X; 3) The role of amino acid X in the function of EF-2; 4) The nature of the diphtheria toxin recognition site on EF-2.
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