Purification and NMR studies of (methyl- sup 13 C)methionine-labeled truncated methionyl-tRNA synthetase

Journal Article · Tue Oct 04 00:00:00 EDT 1988 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00420a052· OSTI ID:6976542

Rosevear, P R ^[1]

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1. Univ. of Texas Medical School, Houston (USA)

A procedure for the rapid purification of a truncated form of the Escherichia coli methionyl-tRNA synthetase has been developed. With this procedure, final yields of approximately 3 mg of truncated methionyl-tRNA synthetase per gram of cells, carrying the plasmid encoding the gene for the truncated synthetase, can be obtained. The catalytic properties of the purified truncated synthetase were found to be identical with those of the native dimeric and trypsin-modified methionyl-tRNA synthetases. A rapid procedure for obtaining milligram quantities of the enzyme is necessary before the efficient incorporation of stable isotopes into the synthetase becomes practical for physical studies. With this procedure, truncated methionyl-tRNA synthetase labeled with (methyl-{sup 13}C)methionine was purified from an Escherichia coli strain auxotrophic for methionine and containing the plasmid encoding the gene for the truncated methionyl-tRNA synthetase. In the absence of ligands, 13 of the 17 methionine residues could be resolved by carbon-13 NMR. Titration of the synthetase, monitoring the chemical shifts of resonances B and M, with a number of amino acid ligands and ATP yielded dissociation constants consistent with those derived from binding and kinetic data, indicating active site binding of the ligands under the conditions of the NMR experiment. The results are consistent with an induced-fit mechanism where portions of the binding site are formed as the various ligands are introduced into the aminoacyl adenylate site.

• OSTI ID: 6976542
• Journal Information: Biochemistry; (USA), Vol. 27:20; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

62 RADIOLOGY AND NUCLEAR MEDICINE
ESCHERICHIA COLI
BIOCHEMICAL REACTION KINETICS
LIGASES
NUCLEAR MAGNETIC RESONANCE
TRANSFER RNA
CROSS-LINKING
CARBON 13
METHIONINE
PLASMIDS
PURIFICATION
AMINO ACIDS
BACTERIA
CARBON ISOTOPES
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CHEMICAL REACTIONS
DRUGS
ENZYMES
EVEN-ODD NUCLEI
ISOTOPES
KINETICS
LIGHT NUCLEI
LIPOTROPIC FACTORS
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
POLYMERIZATION
REACTION KINETICS
RESONANCE
RNA
STABLE ISOTOPES
550601* - Medicine- Unsealed Radionuclides in Diagnostics