Studies on the Polypeptide Elongation Factors from E. coli
VI. Characterization of Sulfhydryl Groups in EF-Tu and EF-Ts
1974 Volume 76 Issue 3 Pages 523-534
Details
1974 Volume 76 Issue 3 Pages 523-534
The properties of sulfhydryl groups in EF-Tu and EF-Ts were investigated by titra-tion with [14C] p-chloromercuribenzoate (pCMB), or 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB), and also by assessing the inhibitory effects of N-ethylmaleimide (NEM) and N-tosyl-L-phenylalanylchloromethane (TPCK) on Tu and Ts activities.
1. EF-Tu contains three sulfhydryl groups, of which two are reactive. One reactive sulfhydryl (SH1) is essential for the binding of GDP or GTP, and is protected by guanine nucleotides in the presence of Mg2+, while the other (SH2) is essential for aminoacyl-tRNA binding, and is protected by aminoacyl-tRNA only when EF-Tu exists as EF-Tu•GTP. The third sulfhydryl group in EF-Tu is non-reactive, probably being buried in the interior of the molecule, and can be titrated only after complete denaturation of the protein.
2. EF-Ts contains two sulfhydryl groups of which one is reactive, while the other is non-reactive in the native configuration of the protein. The former is essential for interaction with EF-Tu and is protected by EF-Tu.
3. Of the five sulfhydryl groups present in EF-Tu•EF-Ts, a complex between EF-Tu and EF-Ts, two are non-reactive, and may correspond to SH3 of EF-Tu and the non-reactive thiol group in EF-Ts. The other three thiols are reactive, but two of them react sluggishly ; SH1 of EF-Tu is protected by EF-Ts, and conversely, the reactive thiol in EF-Ts is protected by EF-Tu.
4. TPCK is found to react selectively with SH2 but not with SH1 when incubated with free EF-Tu in which both SH1 and SH2 are exposed.
5. Kinetic studies indicate that the reactivity of SH2 is higher in EF-Tu•GTP than in EF-Tu•GDP, when measured by titration with [14C]pCMB or by inactivation of the phenylalanyl-tRNA binding activity of Tu with NEM or TPCK. These results suggest the presence of conformational differences between EF-Tu•GTP and EF-Tu•GDP near SH2, i.e., near the site interacting with aminoacyl-tRNA.
