Substitution of aspartic acid-80, a residue involved in coordination of magnesium, weakens the GTP binding and strongly enhances the GTPase of the G domain of elongation factor Tu.
The functional role of Asp80, a residue involved in the coordination of the Mg(2+).guanine nucleotide complex in elongation factor Tu (EF-Tu), has been investigated by its substitution with Asn in the isolated N-terminal domain (G domain). The G domain D80N is characterized by a strong decrease in binding affinity for GTP and magnesium, whereas the affinity for GDP is unchanged. This effect can be mimicked in wild-type G domain by the addition of EDTA. In contrast to this, EDTA does not essentially influence the selective effects of the mutation on the GTP and GDP binding of G domain D80N, indicating that the action of Asp80 is mainly mediated by the GTP-coordinated magnesium ion. The GTPase activity of the G domain D80N is very unstable, but can be markedly stabilized by the addition of glycerol without essentially modifying the specific effects of the mutation. In the absence of glycerol G domain D80N can express a short-lived GTPase activity. The presence of glycerol transforms this evanescent activity into a linear multiple-round activity that under optimal conditions can be almost 2 orders of magnitude higher than the GTPase of wild-type G domain. This enhanced catalytic activity represents the most striking consequence of the mutation and stresses the key role of Asp80 in the GTPase of EF-Tu.(ABSTRACT TRUNCATED AT 250 WORDS)
Harmark
K
K
Centre National de la Recherche Scientifique No. 61840, Laboratoire de Biochimie, Ecole Polytechnique, Palaiseau, France.
Anborgh
P H
PH
Merola
M
M
Clark
B F
BF
Parmeggiani
A
A
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D004492:Edetic Acid / Q000494:pharmacology; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation
D009838:Oligodeoxyribonucleotides; D006153:Guanosine Diphosphate; D001224:Aspartic Acid; D006160:Guanosine Triphosphate; D004492:Edetic Acid; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu; D008274:Magnesium