The 'KMSKS' motif in tyrosyl-tRNA synthetase participates in the initial binding of tRNA(Tyr)

Variants at each position of the 'KMSKS' signature motif in tyrosyl-tRNA sy nthetase have been analyzed to test the hypothesis that this motif is invol ved in catalysis of the second step of the aminoacylation reaction (i.e., t he transfer of tyrosine from the enzyme-bound tyrosyl-adenylate intermediat e to the tRNA(Tyr) substrate). Pre-steady-state kinetic studies show that w hile the rate constants for tyrosine transfer (k(4)) are similar to the wil d-type value for all of the mobile loop variants, the K230A and K233A varia nts have increased dissociation constants (K-d(tRNA) = 2.4 and 1.7 mu M, re spectively) relative to the wild-type enzyme (K-d(tRNA) = 0.39 mu M). In co ntrast, the K-d(tRNA) values for the F231L, G232A, and T234A variants are s imilar to that of the wild-type enzyme. The K-d(tRNA) value for a loop dele tion variant, Delta(227-234), is similar to that for the K230A/K233A double mutant variant (3.4 and 3.0 mu M, respectively). Double mutant free energy cycle analysis indicates there is a synergistic interaction between the si de chains of K230 and K233 during the initial binding of tRNA(Tyr) (Delta D elta G(int) = -0.74 kcal/mol). These results suggest that while the 'KMSKS' motif is important for the initial binding of tRNA(Tyr) to tyrosyl-tRNA sy nthetase, it does not play a catalytic role in the second step of the react ion. These studies provide the first kinetic evidence that the 'KMSKS' moti f plays a role in the initial binding of tRNA(Tyr) to tyrosyl-tRNA syntheta se.