Integrated function of a kinetic proofreading mechanism: double-stage proofreading by isoleucyl-tRNA synthetase.

Experimental measurements for isoleucyl-tRNA synthetase proofreading valyl-tRNAIle in Escherichia coli previously have been incorporated into the conventional Michaelis-Menten model for this system. This model was augmented to include two stages of proofreading--the aminoacyl adenylate and aminoacyl-tRNA stages--and used to predict the values of four additional rate constants that have been determined experimentally. The results suggest that two stages of conventional kinetic proofreading with binding sites designed for isoleucine (the "correct" substrate) are inconsistent with the experimental data, that a double-stage mechanism in which one stage (the "double-sieve") involves a binding site designed for valine (the "incorrect" substrate) and the other involves a binding site designed for isoleucine is consistent with all the experimental data, and that the experimental data are not sufficiently accurate to distinguish the stage at which the double-sieve mechanism operates in vivo. Furthermore, analysis of the model suggests that four parameters have the most questionable values and that experimental refinement of their estimates will be needed to determine which of the two stages involves the double-sieve mechanism.