Active site titration and aminoacyl adenylate binding stoichiometry of aminoacyl-tRNA synthetases

A simple, rapid, and economical procedure is described for the determination of the number of catalytically competent active sites on aminoacyl-tRNA synthetases based on the stoichiometry of aminoacyl adenylate formation. On mixing tRNA synthetase, cognate amino acid, (gamma-32P)ATP, and inorganic p...

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Bibliographic Details
Published in:	Biochemistry (Easton) Vol. 14; no. 1; pp. 1 - 4
Main Authors:	Fersht, Alan R, Ashford, Jeremy S, Bruton, Christopher J, Jakes, Ross, Koch, Gordon L. E, Hartley, Brian S
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 14-01-1975
Subjects:	Amino Acyl-tRNA Synthetases - metabolism Bacillus - enzymology Binding Sites Kinetics Mathematics Molecular Weight Protein Binding Tyrosine
Online Access:	Get full text
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Summary:	A simple, rapid, and economical procedure is described for the determination of the number of catalytically competent active sites on aminoacyl-tRNA synthetases based on the stoichiometry of aminoacyl adenylate formation. On mixing tRNA synthetase, cognate amino acid, (gamma-32P)ATP, and inorganic pyrophosphatase under suitable conditions there is an initial rapid stoichiometric "burst" (rate constant k1) of depletion of ATP as enzyme bound aminoacyl adenylate is formed. There is then an initially linear decrease in ATP concentration as the complex hydrolyzes (with rate constant k2) releasing enzyme to form further adenylate. Provided k2 less than k1 the initial burst gives the stoichiometry of aminoacyl adenylate formation. Complexes which are too unstable to be isolated by the usual gel or nitrocellulose disk filtration procedure may be assayed in this way. This technique has been applied to five highly purified aminoacyl-tRNA synthetases. The tyrosyl-tRNA synthetase from Bacillus stearothermophilus is shown to bind only one aminoacyl adenylate per dimer.
Bibliography:	ark:/67375/TPS-1S5D1NS0-Q istex:AA6C5BBDAF0F7B1824AD595D90B022DC2FA60A64 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0006-2960 1520-4995
DOI:	10.1021/bi00672a001