Structure-function relationships in Escherichia coli translational elongation factor G: modification of lysine residues by the site-specific reagent pyridoxal phosphate

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Abstract
Translational elongation factor G (EF-G) of E. coli was modified with the selective, site specific lysine reagent pyridoxal phosphate (PLP). The reaction results in the modification of a maximum of 12 lysine residues, 1 of which is essential for GTP binding and whose modification is inhibited by the presence of GTP. Formation of a reversible adduct between 2,3-butanedione and an essential arginine similarly located in the GTP binding site also protects EF-G from PLP inactivation, suggesting that these 2 residues are spatially close to each other in the native factor. The essential lysine residue was found in the trypsin-resistant fragment T4 (MW 41,000). In addition to the lysine essential for GTP binding, at least 1 further lysine was important for EF-G function, since GTP-protected, PLP-modified EF-G molecules fully competent in binding to 50S ribosomal subunits showed decreased activity in 50S- and 70S-dependent GTP hydrolysis. A PLP-modified lysine may impair the interaction of the factor with 30S ribosomal subunits. Alternately, a PLP-modified lysine may cause a conformational change of the factor required for the hydrolysis of GTP.

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