Relevance of histidine-84 in the elongation factor Tu GTPase activity and in poly(Phe) synthesis: its substitution by glutamine and alanine.
Substitution of His-84 (-->Gln and -->Ala), a residue of the switch II region of E. coli elongation factor (EF) Tu, hardly affected the binding of GTP or GDP. The activity in poly(Phe) synthesis and GTP hydrolysis of EF-Tu H84Q were both reduced to about 35%, as compared to EF-Tu wt, whereas EF-Tu H84A was inactive in poly(Phe) synthesis but still showed a 10% residual GTPase activity. Phe-tRNAPhe exerted a similar inhibitory effect on the GTPase activity of EF-Tu wt and EF-Tu H84Q while abolishing that of EF-Tu H84A. Ribosomes enhanced the GTPase activity of EF-Tu H84Q, but not that of EF-Tu H84A, on which they even seemed to exert an inhibitory effect. The one-round GTP hydrolysis associated with the EF-TuH84Q-dependent binding of Phe-tRNAPhe to poly(U)-programmed ribosomes was less efficient than with EF-Tu wt. Kirromycin stimulated the GTPase activities of both mutants less than EF-Tu wt. The results of this work do not support a catalytic role of His-84 in the intrinsic GTPase of EF-Tu, but they emphasize the importance of its side-chain for polypeptide synthesis and GTP hydrolysis.
Scarano
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S.D.I. no. 61840 du Centre National de la Recherche Scientifique, Laboratoire de Biochimie, Ecole Polytechnique, Palaiseau, France.
Krab
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Bocchini
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Parmeggiani
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D000409:Alanine; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism*; D005973:Glutamine; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006639:Histidine; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010452:Peptide Biosynthesis; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D010455:Peptides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000378:metabolism
D010455:Peptides; D011994:Recombinant Proteins; D005973:Glutamine; D006153:Guanosine Diphosphate; C012001:polyphenylalanine; D006639:Histidine; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu; D000409:Alanine