Kirromycin, an Inhibitor of Protein Biosynthesis that Acts on Elongation Factor Tu

Abstract

Kirromycin, a new inhibitor of protein synthesis, is shown to interfere with the peptide transfer reaction by acting on elongation factor Tu (EF-Tu). All the reactions associated with this elongation factor are affected. Formation of the EF-Tu·GTP complex is strongly stimulated. Peptide bond formation is prevented only when Phe-tRNA Phe is bound enzymatically to ribosomes, presumably because GTP hydrolysis associated with enzymatic binding of Phe-tRNA Phe is not followed by release of EF-Tu·GDP from the ribosome. This antibiotic also enables EF-Tu to catalyze the binding of Phe-tRNA Phe to the poly(U)·ribosome complex even in the absence of GTP. EF-Tu activity in the GTPase reaction is dramatically affected by kirromycin: GTP hydrolysis, which normally requires ribosomes and aminoacyl-tRNA, takes place with the elongation factor alone. This GTPase shows the same K m for GTP as the one dependent on Phe-tRNA Phe and ribosomes in the absence of the antibiotic. Ribosomes and Phe-tRNA Phe , but not tRNA Phe or Ac-Phe-tRNA Phe , stimulate the kirromycin-induced EF-Tu GTPase. These results indicate that the catalytic center of EF-Tu GTPase that is dependent upon aminoacyl-tRNA and ribosomes is primarily located on the elongation factor. In conclusion, kirromycin can substitute for GTP, aminoacyl-tRNA, or ribosomes in various reactions involving EF-Tu, apparently by affecting the allosteric controls between the sites on the EF-Tu molecule interacting with these components.

Kirromycin‐resistant elongation factor Tu from wild‐type of Lactobacillus brevis

“…The ability of either antibiotic to stimulate the exchange of EF-Tu-bound GDP with unbound GDP at 0°C [1,2] interaction of the drugs with EF-Tu (Lb). Kirromycin and pulvomycin were found to stimulate strongly the nucleotide-binding activity of EF-Tu (Ec) ( fig.2).…”

“…The hydrolysis of GTP involved in the binding of AA-tRNA to ribosomes requires EF-Tu, AA-tRNA, ribosomes and mRNA [8]. In the presence of both pulvomycin or kir romycin, EF-Tu can catalyze the reaction in the absence of the other components [1,2]. When EFTu of L. brevis was used in the antibiotic-dependent hydrolysis of GTP, the two drugs failed to induce GTPase activity even at 100/~M (not shown).…”

“…The antibiotics, kirromycin and pulvomycin, inhibit ptokaryotic protein synthesis by interaction with the elongation factor Tu [1,2]. Mutants of Escherichia coli and Bacillus subtilis selected for resistance to kirromycin have been shown to possess an altered form of EF-Tu [3][4][5].…”

Allosteric changes of the guanine nucleotide site of elongation factor EF‐Tu

“…This antibiotic inhibits the elongation cycle of protein biosynthesis by preventing the release of EF-Tu from the ribosome after hydrolysis of GTP, thus blocking the ribosome on the messenger RNA [9][10][11]. Kirromycin strongly increases the binding affinity of EF-Tu for GTP [12] and gives rise to a turnover GTPase activity of the protein [9].…”

A kirromycin resistant elongation factor EF‐Tu from Escherichia coli contains a threonine instead of an alanine residue in position 375

“…We took advantage of the fact that the antibiotic kirromycin binds to the factor in an 1:1 molar ratio [17]. This binding to EF-Tu • GDP induces an EF-Tu • GTP-like conformation which prevents the release of the factor from the ribosome, thus blocking protein synthesis [ 15,18,19].…”