Stereochemistry of the elongation factor Tu X GTP complex.

The geometry of the Me2+. GTP complex at the active site of EF-Tu from Bacillus stearothermophilus has been investigated using thiophosphate analogs of GTP to inhibit the kirromycin-induced GTPase reaction at 60 mM NH4Cl. There is no reversed selectivity for the diastereomers (Rp and Sp) of guanosine 5'-O-(1-thiotriphosphate) (GTP[alpha S]) on replacing Mg2+ by Cd2+, so that the observed specifity for the Sp isomer must be due to an interaction of the pro-R oxygen of the alpha-phosphate group with the protein. With the diastereomers of GTP[beta S] low specifity for the Rp isomers is seen in the presence of Mg2+. Moreover, both isomers are very weakly bound. In contrast, substitution of Mg2+ by Cd2+ results in a high specifity for the Sp isomer, and this is then recognized as well as Cd X GTP. These results indicate that in the EF-Tu X Me2+ X GTP complex, the pro-S oxygen of the beta-phosphate group is bound to the metal ion and the pro-R oxygen to the protein. GTP[gamma S] is a good analog of GTP regardless of the nature of the metal ion, suggesting that not all of the oxygens of the gamma-phosphate are involved in interactions to metal ion and protein. The thiophosphate analogs of GTP were also tested for their efficiency in ternary complex formation with EF-Tu and aminoacyl-tRNA and in the physiological GTPase of EF-Tu. The stereochemistry of the GTP binding site on EF-Tu in all three systems is found to be very similar.