Selective photooxidation of histidine residues in polypeptide chain elongation factor Tu from E. coli.
When EF-Tu was photooxidized for 20 min at 0 degrees C in the presence of 10 microM GDP and 5 microM rose bengal, the activity to promote the binding of [14C]Phe-tRNA to ribosomes was rapidly lost, while the activity to bind [3H]GDP remained intact. The activity of EF-Tu to interact with Phe-tRNA and ribosomes, as assessed by protection of [14C]Phe-tRNA against RNase A digestion and by methanol-induced uncoupled GTPase activity, respectively, was also inactivated under the above conditions. It was found, however, that these activities were fully protected in the presence of aminoacyl-tRNA and GTP, indicating that the active site(s) of EF-Tu for interaction with aminoacyl-tRNA and ribosomes could be protected against photooxidation in the ternary aminoacyl-tRNA . EF-Tu . GTP complex. Comparison of the amino acid composition of EF-Tu photooxidized in the form of EF-Tu . GDP with that of the intact EF-Tu revealed that only 1.4 residues of histidine were damaged. On the other hand, no histidine residue was lost when EF-Tu was oxidized in the presence of both aminoacyl-tRNA and GTP. The photooxidized EF-Tu . GDP was then partially degraded with trypsin and each of the resulting tryptic fragments, D, B, and C (Arai, Nakamura, Arai, Kawakita, and Kaziro (1976) J. Biochem. 79, 69-83), was analyzed for histidine content. The results indicated that fragments B, C, and D had lost 0.7, 0.5, and 0.2 residues of histidine, respectively. Since fragment B contains the cysteine residue which is essential for interaction with aminoacyl-tRNA and ribosomes, the above results suggest that a histidine residue in fragment B may also play an essential role in the interaction with aminoacyl-tRNA and ribosomes.
D000596:Amino Acids / Q000032:analysis; D004926:Escherichia coli; D006153:Guanosine Diphosphate / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D010084:Oxidation-Reduction; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010777:Photochemistry; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012395:Rose Bengal / Q000378:metabolism; D013997:Time Factors
D000596:Amino Acids; D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; D006153:Guanosine Diphosphate; D012395:Rose Bengal; D006639:Histidine; D010444:Peptide Elongation Factor Tu