Student Theses and Dissertations
Date of Award
1968
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Fritz Lipmann
Keywords
aminoacyl-tRNA synthetase, amino acids, hydroxamate assay, threonyl-tRNA synthetase, enzyme protection, protein synthesis
Abstract
Amino acids are activated as aminoacyladenylates which remain bound to the aminoacyl-tRNA synthetases that catalyze their formation. The activated amino acids are then esterified to specific transfer RNA molecules. By this reaction sequence, the specificity and energetics necessary for polypeptide synthesis are conferred upon the amino acids. The first method of determining acyl group activation was the hydroxamate assay. The activated amino acids can be trapped as the stable amino acid hydroxamates by carrying out the activation reactions in high concentrations of hydroxylamine. This thesis comprises a study of the hydroxamate reaction of the aminoacyl-tRNA synthetases. Each of fourteen partially purified aminoacyl-tRNA synthetases from E. coli was assayed for the rates of formation of amino acid hydroxamate and aminoacyl-tRNA. These relative rates of amino acid hydroxamate formation were found to vary over a sixtyfold range. The methionyl- and aromatic aminoacyl-tRNA synthetases catalyze hydroxamate formation at rates comparable to the rates of acylation of tRNA. In descending order, Lys-, Ala-, Ile-, Gly-, Cys-, Leu-, Ser-, and Val-tRNA synthetases catalyze hydroxamate formation at rates below their respective rates of aminoacyl-tRNA formation. This variation is irrespective of the sources of the enzymes, and it is interpreted to reflect the differential protection that the aminoacyl-tRNA synthetases afford their respective aminoacyladenylates. The most extreme case in this regard is the threonyl-tRNA synthetase, which does not catalyze threonine hydroxamate formation. Therefore, this enzyme was studied in detail. The E. coli threonyl-tRNA synthetase was purified 320 fold. The enzyme activates threonine, as determined by the ATP-PPi exchange reaction, and it readily esterifies threonine to tRNA. Only if tRNA supplements the threonyl-tRNA synthetase hydroxamate assay does threonine hydroxamate continuously form via the nonenzymatic reaction of hydroxylamine with the enzymatically produced threonyl-tRNA. The threonyladenylate-enzyme complex was isolated. The complex transfers threonine to tRNA. This transfer requires Mg++, although Ca++ or Mn++ can substitute for Mg++. The optimal Mg++ concentration for this transfer reaction is 2 mM whereas the optimum for the overall reaction is 10 mM. PCMBS inhibits the transfer reaction but NEM does not. The complex spontaneously hydrolyzes at 30°, pH 7, with a half life of 29 minutes. When the isolated complex is incubated with hydroxylamine, it immediately disintegrates. Threonine and not threonine hydroxamate is the reaction product. Hydrolysis instead of hydroxaminolysis occurs. Therefore, the enzyme is converted from a synthetase to a hydrolase in the presence of hydroxylamine. For comparison, the E. coli phenylalanyl-tRNA synthetase was studied. This enzyme catalyzes phenylalanine hydroxamate formation at a rate within 20% of the rate of phenylalanyl-tRNA formation. This enzyme is also inhibited by PCMBS. The phenylalanyladenylate-enzyme complex was isolated. It requires Mg++ for transfer of phenylalanine to tRNA. It is fourfold less stable to spontaneous hydrolysis than is the threonyladenylate-enzyme complex under the same conditions. When incubated in hydroxylamine, phenylalanyladenylate-enzyme complex disintegrates. In contrast to the case of threonyladenylate-enzyme complex, phenylalanine hydroxamate is the reaction product. As a result of these studies, it is proposed that the differential rates of amino acid hydroxamate formation by the various aminoacyl-tRNA synthetases are due to two factors: the sequestering of the aminoacyladenylates by the enzymes, and the conversion of the enzymes from synthetases to hydrolases. The latter feature is explicit in threonyl-tRNA synthetase. These may be protective mechanisms by which synthetases prevent nucleophilic attack by molecules other than the natural substrates. In this manner, false transfer reactions, which would prove lethal to the organism, are avoided.
License and Reuse Information
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Recommended Citation
Hirsch, David Ian, "The Hydroxamate Reaction of Aminoacyl-tRNA Synthetases" (1968). Student Theses and Dissertations. 566.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/566
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy