Effect of Kirromycin on Elongation Factor Tu. Location of the Catalytic Center for Ribosome . Elongation-Factor-Tu GTPase Activity on the Elongation Factor

Abstract

Elongation factor Tu (EF-Tu) is the target of kirromycin, an inhibitor of protein biosynthesis that enables EF-Tu to catalyze GTP hydrolysis in the absence of ribosomes and aminoacyl-tRNA. This GTPase activity conserves many of the characteristics of the physiological one, which is triggered by ribosomes plus aminoacyl-tRNA. Both activities have the same substrate specificity and are affected by aminoacyl-tRNA, but not by tRNA or N-acetylaminoacyl-tRNA. With kirromycin, aminoacyl-tRNA changes only the V of the reaction without affecting the Km for GTP, which remains the same as the one determined in the presence of aminoacyl-tRNA plus ribosomes without the antibiotic. Addition of ribosomes does not affect the course of temperature-dependence of the kirromycin-induced GTPase activity. Our results show that the catalytic center for GTP hydrolysis, associated with the enzymic binding of aminoacyl-tRNA to ribosome, is located primarily on the elongation factor and not on the ribosome. Kirromycin binds to EF-Tu in a molar ratio of 1:1 and competes with elongation factor Ts (EF-Ts) for this binding, favoring the state of dissociation of EF-Tu and EF-Ts, probably by inhibiting their reassociation. All the partial reactions involved in the formation of the ternary complex (aminoacyl-tRNA · EF-Tu · GTP) are affected by the antibiotic, which stimulates strongly EF-Tu affinity for GTP and consequently the GDP-GTP exchange reaction with EF-Tu · GDP. Kirromycin activates the EF-Tu site for aminoacyl-tRNA even in the presence of GDP, abolishing the requirement of GTP for formation of the aminoacyl-tRNA · EF-Tu complex. Our results show that kirromycin does not interact directly with the EF-Tu sites for guanine nucleotides, aminoacyl-tRNA and ribosomes. Its action, which allosterically relieves tight mutual control mechanisms, appears to be directed towards a critical region of the EF-Tu molecule responsible for the regulation of the sites involved in the elongation process.