The geometry of the Me2+ . G T P complex at the active site of EF-Tu from Bacillus stearothermophilus has been investigated using thiophosphate analogs of G T P to inhibit the kirromycin-induced GTPase reaction at 60 mM NH4CI. There is no reversed selectivity for the diastereomers ( R , and S,) of guanosine 5'-0-(1 -thiotriphosphate) (GTP[cnS]) on replacing Mg2+ by C d 2 + , so that the observed specifity for the S, isomer must be due to an interaction of thepro-R oxygen of the x-phosphate group with the protein. With the diastereomers of GTP[PS] low specifity for the R, isomers is seen in the presence of ME'+. Moreover, both isomers arc very weakly bound. In contrast, substitution of Mg2+ by Cd2+ results in a high specifity for the S, isomer, and this is then recognized as well as Cd . GTP. These results indicate that in the EF-Tu . Me2+ . GTP complex, the pro-S oxygen of the /I-phosphate group is bound to the metal ion and thepro-R oxygen to the protein. GTP[.;S] is a good analog of G T P regardless of the nature of the metal ion, suggesting that not all of the oxygcns of the y-phosphate are involved in interactions to metal ion and protein. The thiophosphate analogs of G T P were also tested for their efficicncy in ternary complex formation with EF-Tu and aminoacyl-tRNA and in the physiological GTPase of EF-Tu. The stereochemistry ofthe GTP binding site on EF-Tu in all three systems is found to be very similar.During the elongation cycle of protein biosynthesis, the codon-directed binding of aminoacyl-tRNA to the ribosome takes place via an elongation factor Tu . G T P . aminoacyltRNA complex (for reviews see [I ,2]). This EF-Tu-mediated binding process also involves the factor-mediated hydrolysis of GTP to G D P and Pi. The triphosphate hydrolysis is now believed to be required for proof-reading, an energy-requiring mechanism, which leads to an abortive binding of improper aminoacyl-tRNA and, in the case of the correct substrate, to a release of EF-Tu . G D P from the ribosome and to incorporation into the polypeptide chain of the aminoacyl group [3,4].EF-Tu as a G D P complex has a low affinity and as a GTP complex a high affinity for aminoacyl-tRNA [5]. It is now well documented that this protein carries the active site of the GTPase reaction, although uncoupled from protein biosynthesis the reaction rate is very low, if not immeasurable [6]. In the presence of kirromycin it can be induced to show reasonable turnover activity; this has enabeled Parmeggiani and coworkers to study many of the kinetic and thermodynamic parameters of this reaction [7,8]. Using thiophosphatc analogs of GTP, we have studied the active-site structure of EF-Tu. GTP and compared this with rcsults obtained carlier using the corresponding analogs of G D P [9] to obtain insight into the stereochemical course of the GTPase reaction and to -~/,hi.c,f.rtrtrorz,s. EF-Tu, if not indicated otherwise, is the protein synthcsis elongation factor Tu from Brrci1lu.s sterurother/nophilus; EF-T is the complex between EF-Tu and protein synthesis el...
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