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 synthetase have been analyzed to test the hypothesis that this motif is involved in catalysis of the second step of the aminoacylation reaction (i.e., the transfer of tyrosine from the enzyme-bound tyrosyl-adenylate intermediate to the tRNA(Tyr) substrate). Pre-steady-state kinetic studies show that while the rate constants for tyrosine transfer (k(4)) are similar to the wild-type value for all of the mobile loop variants, the K230A and K233A variants have increased dissociation constants (K(d)(tRNA)( )()= 2.4 and 1.7 microM, respectively) relative to the wild-type enzyme (K(d)(tRNA)( )()= 0.39 microM). In contrast, the K(d)(tRNA) values for the F231L, G232A, and T234A variants are similar to that of the wild-type enzyme. The K(d)(tRNA) value for a loop deletion variant, Delta(227-234), is similar to that for the K230A/K233A double mutant variant (3.4 and 3.0 microM, respectively). Double mutant free energy cycle analysis indicates there is a synergistic interaction between the side chains of K230 and K233 during the initial binding of tRNA(Tyr) (DeltaDeltaG(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 synthetase, it does not play a catalytic role in the second step of the reaction. These studies provide the first kinetic evidence that the 'KMSKS' motif plays a role in the initial binding of tRNA(Tyr) to tyrosyl-tRNA synthetase.
Xin
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Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center in Shreveport, Shreveport, Louisiana 71130, USA.
Li
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D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D007700:Kinetics; D009154:Mutation; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry*; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry*
D012346:RNA, Transfer, Amino Acyl; D014443:Tyrosine; D000604:Amino Acyl-tRNA Synthetases