Abstract
The stereochemical course of the GTPase of elongation factor Tu from Escherichia coli has been determined by making use of the reaction dependent on antibiotic X5108 (an N-methylated derivative of kirromycin). Guanosine 5'-(gamma-thio)triphosphate stereospecifically labeled with 17O and 18O in the gamma-position was hydrolyzed in the presence of elongation factor Tu and X5108. The configuration of the product, inorganic [16O, 17O, 18O]thiophosphate was analyzed by 31P NMR after its stereospecific incorporation into adenosine 5'-(beta-thio)triphosphate. The analysis showed that the hydrolysis proceeds with inversion of configuration at the transferred phosphorus, implying that there is not a phosphoenzyme intermediate. Incubation of [beta gamma-18O, gamma-18O3]GTP with elongation factor Tu leaves the 18O-labeling unaltered, as shown by 31P NMR. No exchange of oxygens with water nor beta gamma-beta positional isotope exchange occurs, implying that not even transient cleavage can occur with the elongation factor alone. Only on interaction with X5108, kirromycin, or ribosomes does the cleavage occur, most likely by a single step, in-line transfer of the terminal phosphorus from GDP to a water oxygen. These properties of the GTP hydrolysis mechanism of elongation factor Tu are similar to those of elongation factor G.
Highlights
The stereochemical course of the GTPaseof elonga- produce the necessary chirality:2 tion factor Tu from Escherichia coli has been determinedbymaking use ofthereactiondependent on antibiotic X5108.Guanosine 5’-(y-thio)triphosphatsetereospecifically labeled with I7O and “0 in the y-position was
In the elongation cycle of protein biosynthesis in prokaryotic organisms, the elongation factor EF-TU’ mediates the binding of aminoacyl-tRNA to the A site of ribosomes,a process which precedes the formation of each new peptide bond. This binding of aminoacyl-tRNA is accompanied by GTP hydrolysis (Miller and Weissbach, 1977; Thompson et al, 1980) the relationship between these two steps is not yetclear
As part of an investigationof the elementary processes involved in the elongation cycle, we have studied the mechanism of GTP hydrolysis by EF-Tu using oxygen isotopes, and in particular we have determined the stereochemical course of this GTPase
Summary
Overall retention is explained change occurs, implying thant ot even transient cleav- most by two steps: the formation anbdreakdown of a age can occurwith the elongationfactor alone.Only on phosphorylated intermediate This stereochemical technique interaction with X5108, kirromycin, or ribosomesdoes has been applied previously to three ATPases,myosin, mitothe cleavage occur, mostlikely by a single step, in-line transfer of theterminalphosphorusfromGPP to a water oxygen. In the elongation cycle of protein biosynthesis in prokaryotic organisms, the elongation factor EF-TU’ mediates the binding of aminoacyl-tRNA to the A site of ribosomes,a process which precedes the formation of each new peptide bond This binding of aminoacyl-tRNA is accompanied by GTP hydrolysis (Miller and Weissbach, 1977; Thompson et al, 1980) the relationship between these two steps is not yetclear. The rateof hydrolysis was calculated from the linear portion of the reaction time course
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