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Site‐directed mutagenesis of Thermus thermophilus EF‐Tu: the substitution of threonine‐62 by serine or alanine
Author(s) -
Reza Ahmadian M.,
Kreutzer Roland,
Blechschmidt Bernd,
Sprinzl Mathias
Publication year - 1995
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(95)01354-7
Subject(s) - thermus thermophilus , gtp' , ef tu , gtpase , biochemistry , threonine , site directed mutagenesis , alanine , serine , biology , chemistry , ribosome , escherichia coli , amino acid , phosphorylation , rna , enzyme , mutant , gene
The invariant threonine‐62, which occurs in the effector region of all GTP/GDP‐binding regulatory proteins, was substituted via site‐directed mutagenesis by alanine and serine in the elongation factor Tu from Thermus thermophilus . The altered proteins were overproduced in Escherichia coli , purified and characterized. The EF‐Tu T62S variant had similar properties with respect to thermostability, aminoacyl‐tRNA binding, GTPase activity and in vitro translation as the wild‐type EF‐Tu. In contrast, EF‐Tu T62A is severely impaired in its ability to sustain polypeptide synthesis and has only very low intrinsic and ribosome‐induced GTPase activity. The affinity of aminoacyl‐tRNA to the EF‐Tu T62A·GTP complex is almost 40 times lower as compared to the native EF‐Tu·GTP. These observations are in agreement with the tertiary structure of EF‐Tu·GTP, in which threonine‐62 is interacting with the Mg 2+ ion, γ‐phosphate of GTP and a water molecule, which is presumably involved in the GTP hydrolysis.