Raimo G, Masullo M, Bocchini V
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Italia.
J Biol Chem. 1995 Sep 8;270(36):21082-5. doi: 10.1074/jbc.270.36.21082.
The elongation factor 2 from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-2) binds [3H]GDP at 1:1 molar ratio. The bound [3H]GDP is displaced by GTP or its nonhydrolyzable analogue guanyl-5'-yl imidodiphosphate (Gpp(NH)p) but not by ATP, thus indicating that only the two guanosine nucleotides compete for the same binding site. The affinity of SsEF-2 for [3H]GDP is higher than that for GTP and Gpp(NH)p. On the contrary, in the presence of ribosomes the affinity of SsEF-2 for GDP is lower than that for Gpp(NH)p. SsEF-2 is endowed with an intrinsic hardly detectable GTPase activity that is stimulated by ribosomes up to 2000-fold. The ribosome-stimulated SsEF-2 GTPase (GTPaser) reaches a maximum at pH 7.8 and is not affected by ATP but is competitively inhibited by either GDP or Gpp(NH)p. Both Km for [gamma-32P]GTP and kcat of GTPaser increase with increasing temperature, and the highest catalytic efficiency is reached at 80 degrees C. The ADP-ribosylation of SsEF-2 does not significantly affect either the binding of GDP and GTP or the kinetics of the GTPaser. A hypothesis on the stimulation by ribosome of SsEF-2 GTPase is proposed.
嗜热嗜酸古菌嗜热栖热菌（Sulfolobus solfataricus）的延伸因子2（SsEF-2）以1:1的摩尔比结合[3H]GDP。结合的[3H]GDP可被GTP或其不可水解类似物鸟苷-5'-亚基亚氨基二磷酸（Gpp(NH)p）取代，但不能被ATP取代，这表明只有这两种鸟苷酸竞争同一个结合位点。SsEF-2对[3H]GDP的亲和力高于对GTP和Gpp(NH)p的亲和力。相反，在核糖体存在的情况下，SsEF-2对GDP的亲和力低于对Gpp(NH)p的亲和力。SsEF-2具有内在的几乎难以检测到的GTPase活性，该活性可被核糖体刺激高达2000倍。核糖体刺激的SsEF-2 GTPase（GTPaser）在pH 7.8时达到最大值，不受ATP影响，但被GDP或Gpp(NH)p竞争性抑制。[γ-32P]GTP的Km和GTPaser的kcat均随温度升高而增加，在80℃时达到最高催化效率。SsEF-2的ADP核糖基化对GDP和GTP的结合或GTPaser的动力学均无显著影响。本文提出了关于核糖体对SsEF-2 GTPase刺激作用的假说。
