Abrahams J P, Acampo J J, Kraal B, Bosch L
Department of Biochemistry, Leiden University, The Netherlands.
Biochimie. 1991 Jul-Aug;73(7-8):1089-92. doi: 10.1016/0300-9084(91)90150-y.
The turnover of EF-Tu.GTP on poly-U programmed ribosomes was measured both in the presence and in the absence of N-acetylated Phe-tRNA(Phe) at the P-site. The reaction was uncoupled from protein synthesis by omitting Phe-tRNA(Phe) at the A-site. In this reaction, the ribosome can be considered as an enzyme catalysing the transition of EF-Tu.GTP to EF-Tu.GTP. A constant EF-Tu.GTP concentration is maintained by regenerating GDP to GTP at the expense of phosphoenolpyruvate by pyruvate kinase. The rate constants are determined using a procedure which corrects for the reduction in specific activity of GTP due to regeneration of the nucleotide. Ribosomes with an occupied P-site are more efficient in stimulating the GTPase of EF-Tu.GTP than ribosomes with an empty P-site. The data suggest that this is mainly caused by an increased affinity of EF-Tu.GTP for ribosomes with a filled P-site rather than by an enhanced reactivity of the GTPase centre.
在P位点存在和不存在N - 乙酰化苯丙氨酰 - tRNA（Phe）的情况下，均测定了多聚 - U程序化核糖体上EF - Tu·GTP的周转情况。通过省略A位点的苯丙氨酰 - tRNA（Phe），使该反应与蛋白质合成解偶联。在该反应中，核糖体可被视为一种催化EF - Tu·GTP向EF - Tu·GDP转变的酶。通过丙酮酸激酶以磷酸烯醇式丙酮酸为代价将GDP再生为GTP，从而维持EF - Tu·GTP浓度恒定。使用一种校正由于核苷酸再生导致的GTP比活性降低的程序来确定速率常数。占据P位点的核糖体比空P位点的核糖体在刺激EF - Tu·GTP的GTP酶活性方面更有效。数据表明，这主要是由于EF - Tu·GTP对占据P位点的核糖体的亲和力增加，而不是由于GTP酶中心的反应性增强。
