Studies on the Polypeptide Elongation Factors from E. coli

Free EF-Tu is extremely unstable, being inactivated almost completely at 40°C within 5 mm, but it is markedly stabilized by GDP, EF-Ts, GTP, 5′-guanylylmethyl-enediphosphonate (Gpp(CH2)p) and Phe-tRNA plus GTP. EF-Ts is heat stable, full activity being retained after heating for 5 mm at 60°C. The pH stability of EF-Tu, EF-Ts, the complex of EF-Tu and GDP(Tu.GDP), and the complex of EF-Tu and EF-Ts(Tu.Ts) is also described. 2. The optimal pH for the Tu.GDP ⇌3HGDP exchange reaction catalyzed by EF-Ts is around pH 8.0. 3. Dissociation constants of Tu.GDP in the presence and absence of Mg2+ ions are 4.9×10−9M, and 2.2×10−6 respectively. Those of Tu.Ts, Tu and Tu.Gpp (CH2)p are 7.7′10−9M 3.6′10−7 M, and 2.2×10−6 respectively. Ternary complexes, (Phe-tRNA).Tu.GTP and (Phe-tRNA).Tu.Gpp(CH2 dissociate into Phe-tRNA and Tu. nucleotide complexes with dissociation constants of 7.2′10−7 M and 2.5×10−7 M, respectively. 4. The first order rate constant for dissociation (k−1 of Tu.GDP is 3.4×10−4 sec−1 at 0°C. The second order rate constant for association (k+1 of EF-Tu and GDP is 3.1×105 at 0°C. Similarly defined rate constants k−1 and k+1 of Tu.GTP are 5.0×10−2 sec−1 and 1.1×105 respectively. Both the k+1 and k−1 values are markedly affected by temperature. 5. The ratio of k−1 to k+1 agrees well with the dissociation constants of Tu.GDP and Tu.The dissociation constants are little affected by temperature.