Identification of the N-tosyl-L-phenylalanyl chloromethylketone modification site in Thermus thermophilus elongation factor Tu.
EF-Tu from Thermus thermophilus was first labelled with N-[14C]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 of 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.
Peter
M E
ME
Laboratorium für Biochemie, Universität Bayreuth, FRG.
Brockmöller
J
J
Jonák
J
J
Sprinzl
M
M
D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism*; D001665:Binding Sites; D007700:Kinetics; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013824:Thermus / Q000378:metabolism*; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D010446:Peptide Fragments; D014108:Tosylphenylalanyl Chloromethyl Ketone; D010444:Peptide Elongation Factor Tu