Wohlgemuth Ingo, Brenner Sibylle, Beringer Malte, Rodnina Marina V
Institute of Physical Biochemistry, University of Witten/Herdecke, Stockumer Strasse 10, D-58448 Witten, Germany.
J Biol Chem. 2008 Nov 21;283(47):32229-35. doi: 10.1074/jbc.M805316200. Epub 2008 Sep 22.
The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s(-1), regardless of the tRNA identity. The reactivity decreased in the order Lys = Arg > Ala > Ser > Phe = Val > Asp >> Pro, with Pro being by far the slowest. However, when Phe-tRNA(Phe) was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was approximately 7 s(-1), which corresponds to the rate of binding of Phe-tRNA(Phe) to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyl-tRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.
核糖体催化P位点的肽基-tRNA与A位点的氨酰-tRNA之间形成肽键。在此，我们表明P位点肽基-tRNA中C末端氨基酸残基的性质强烈影响肽基转移的速率。取决于肽基-tRNA的C末端氨基酸，与小的A位点底物嘌呤霉素的反应速率在100至0.14 s(-1)之间变化，与tRNA的身份无关。反应活性按以下顺序降低：赖氨酸=精氨酸>丙氨酸>丝氨酸>苯丙氨酸=缬氨酸>天冬氨酸>>脯氨酸，其中脯氨酸是迄今为止最慢的。然而，当苯丙氨酰-tRNA(Phe)用作A位点底物时，与任何肽基-tRNA形成肽键的速率约为7 s(-1)，这与苯丙氨酰-tRNA(Phe)与A位点的结合速率（容纳）相对应。由于容纳是肽键形成的限速步骤，所以对于所有肽基-tRNA，反应速率是一致的，而与内在化学反应活性的变化无关。另一方面，以脯氨酸结尾的肽基-tRNA的反应速率增加50倍表明A位点的全长氨酰-tRNA极大地加速了肽键的形成。
