Delayed release of inorganic phosphate from elongation factor Tu following GTP hydrolysis on the ribosome.

The dissociation of inorganic phosphate (P(i)) following GTP hydrolysis is a key step determining the functional state of many GTPases. Here, the timing of P(i) release from elongation factor Tu (EF-Tu) and its implications for the function of EF-Tu on the ribosome were studied by rapid kinetic techniques. It was found that P(i) release from EF-Tu is >20-fold slower than GTP cleavage and limits the rate of the conformational switch of EF-Tu from the GTP- to the GDP-bound form. The point mutation Gly94Ala in the switch 2 region of EF-Tu abolished the delay in P(i) release, suggesting that P(i) release is controlled by the mobility of the switch 2 region with Gly94 acting as a pivot. The rate of P(i) release or the conformational switch of EF-Tu does not affect the selection of aminoacyl-tRNA on the ribosome. Rather, the slow P(i) release may be a consequence of the tight interaction of the switch regions of EF-Tu with the gamma-phosphate and the ribosome in the GTPase activated state of the factor.