PMID:10529173
| Citation |
Krab, IM and Parmeggiani, A (1999) Mutagenesis of three residues, isoleucine-60, threonine-61, and aspartic acid-80, implicated in the GTPase activity of Escherichia coli elongation factor Tu. Biochemistry 38:13035-41 |
|---|---|
| Abstract |
The properties of variants of elongation factor (EF) Tu mutated at three positions implicated in its GTPase activity are presented. Mutation I60A, which reduces one wing of a "hydrophobic barrier" screening off the nucleophilic water molecule found at the GTP gamma-phosphate, causes a reduction of the intrinsic GTPase activity contrary to prediction and has practically no influence on other properties. Mutation D80N, which in the isolated G-domain of EF-Tu caused a strong stimulation of the intrinsic GTPase, reduces this activity in the intact molecule. However, whereas for wild-type EF-Tu complex formation with aa-tRNA reduces the GTPase, EF-Tu[D80N] shows a strongly increased activity when bound to Phe-tRNA. Moreover, ribosomes or kirromycin can stimulate its GTPase up to the same level as for wild-type. This indicates that a local destabilization of the magnesium binding network does not per se cause an increased GTPase but does affect its tight regulation. Interestingly, mutant D80N sequestrates EF-Ts by formation of a more stable complex. Substitutions T61A and T61N induce low intrinsic GTPase, and the stimulation by ribosome is less for T61A than for T61N but still detectable, while kirromycin stimulates the GTPase of both mutants equally. This provides more evidence that stimulation by kirromycin and ribosomes follows a different mechanism. The functional implications of these mutations are discussed in the context of a transition state mechanism for catalysis. An alternative structural explanation for the strong conservation of Ile-60 is proposed. |
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| Keywords |
Aspartic Acid/genetics; Binding Sites/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Esters; GTP Phosphohydrolases/genetics; GTP Phosphohydrolases/metabolism; Guanosine Diphosphate/metabolism; Guanosine Triphosphate/metabolism; Isoleucine/genetics; Magnesium/chemistry; Magnesium/metabolism; Models, Molecular; Mutagenesis, Site-Directed; Peptide Elongation Factor Tu/antagonists & inhibitors; Peptide Elongation Factor Tu/biosynthesis; Peptide Elongation Factor Tu/genetics; Peptide Elongation Factor Tu/metabolism; Peptides/chemical synthesis; Pyridones/chemistry; RNA, Transfer, Phe/metabolism; Recombinant Proteins/antagonists & inhibitors; Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Ribosomes/metabolism; Threonine/genetics |
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