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Identification of the N ‐tosyl‐L‐phenylalanyl chloromethylketone modification site in Thermus thermophilus elongation factor Tu
Author(s) -
Peter Marcus E.,
Brockmöller Jürgen,
Jonák Jiří,
Sprinzl Mathias
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)81538-8
Subject(s) - thermus thermophilus , tosyl , chemistry , trypsin , cleavage (geology) , peptide , stereochemistry , ef tu , peptide bond , conformational change , biochemistry , transfer rna , enzyme , biology , rna , paleontology , escherichia coli , fracture (geology) , gene
EF‐Tu from Thermus thermophilus was first labelled with N ‐[ 14 C]tosyl‐L‐phenylalaninechloromethylketone and then cleaved by the combined action of CNBr and trypsin. The resulting peptides were separated by reversed‐phase HPLC. Analysis of the isolated, labelled peptide led to the identification of a sequence which was identical to residues 76–88 in T. thermophilus EF‐Tu. The TPCK reactive site is at Cys‐82. Kinetic measurements f the incorporation of TPCK into native EF‐Tu and EF‐Tu nicked at position Arg‐59 were performed. The results provide evidence that the cleavage of the peptide bond between Arg‐59 and Gly‐60 does not lead to a dramatic conformational change of EF‐Tu at the aa‐tRNA binding site.