Kinetic analysis of the isoleucyl‐tRNA synthetase mechanism: the next reaction cycle can start before the previous one ends

Abstract

Aminoacyl‐tRNA synthetases join correct amino acids to their cognate tRNA at the start of the protein synthesis. Through the kinetic analysis, it is possible to estimate how their functional details correspond to the known structural features. Kinetic analysis of the isoleucyl‐tRNA synthetase (IleRS) from Escherichia coli was accomplished. Sixteen different steady‐state two‐ligand experiments were statistically analysed simultaneously so that the same rate equations and same rate and dissociation constants applied to all experiments. The so‐called rapid equilibrium segments procedure was used to derive the rate equations. The final best‐fit mechanism included the normal activation and transfer steps, and reorganization of the steps between them and after the transfer step. In addition, the analysis strongly suggested an additional activation step, formation of a new isoleucyl‐AMP before the isoleucyl‐tRNA was freed from the enzyme. The removal of Ile‐tRNA was possible without the formation of Ile‐AMP if both isoleucine and ATP were bound to the E‐Ile‐tRNA complex, but this route covered only 11% of the total formation of Ile‐tRNA. In addition to the Mg 2+ in MgATP or MgPP i , only two tRNA‐bound Mg 2+ were required to explain the magnesium dependence in the best‐fit mechanism. The first Mg 2+ could be present in all steps before the second activation and was obligatory in the first reorganizing step and transfer step. The second Mg 2+ was present only at the transfer step, whereas elsewhere it prevented the reaction, including the activation reactions. Chloride inhibited the IleRS reaction, while 100 m m KCl caused 50% inhibition if the ionic strength was kept constant with K‐acetate. The (tRNA) value was increased from 0.057 to 1.37 μ m when the KCl concentration was increased from 0 to 200 m m . The total rate equation helps to understand the reaction route and how the simultaneous presence of Ile‐tRNA and Ile‐AMP can cause new possibilities to proofreading mechanisms of this enzyme. Enzyme Isoleucyl‐tRNA synthetase ( EC 6.1.1.5 )

Partitioning of the initial catalytic steps of leucyl-tRNA synthetase is driven by an active site peptide-plane flip

“…This in turn indicates that the mobility of CP1 hairpin influenced by both L550 and E169 appears to be important for the disassociation of the final product. Detailed studies of IleRS, an enzyme belonging to the same aaRS subclass as LeuRS have demonstrated that the rebinding of Ile and ATP, or formation of the catalytic cycle intermediate Ile-AMP, occurs first and promotes the release of Ile-tRNA Ile 50 – 52 . Similarly, we hypothesize that LeuRS may also start the next cycle of Leu activation leading to closure of the CP1 hairpin which promotes the dissociation of Leu-tRNA Leu (Fig.…”