Site-directed mutagenesis reveals transition-state stabilization as a general catalytic mechanism for aminoacyl-tRNA synthetases.
Some aminoacyl-tRNA synthetases of almost negligible homology do have a small region of similarity around four-residue sequence His-Ile(or Leu or Met)-Gly-His(or Asn), the HIGH sequence. The first histidine in this sequence in the tyrosyl-tRNA synthetase, His-45, has been shown to form part of a binding site for the gamma-phosphate of ATP in the transition state for the reaction as does Thr-40. Residue His-56 in the valyl-tRNA synthetase begins a HIGH sequence, and there is a threonine at position 52, one position closer to the histidine than in the tyrosyl-tRNA synthetase. The mutants Thr----Ala-52 and His----Asn-56 have been made and their complete free energy profiles for the formation of valyl adenylate determined. Difference energy diagrams have been constructed by comparison with the reaction of wild-type enzyme. The difference energy profiles are very similar to those for the mutants Thr----Ala-40 and His----Asn-45 of the tyrosyl-tRNA synthetase. Thr-52 and His-56 of the valyl-tRNA synthetase contribute little binding energy to valine, ATP, and Val-AMP. Instead, the wild-type enzyme binds the transition state and pyrophosphate some 6 kcal/mol more tightly than do the mutants. Preferential transition-state stabilization is thus an important component of catalysis by the valyl-tRNA synthetase. Further, by analogy to the tyrosyl-tRNA synthetase, the valyl-tRNA synthetase has a binding site for the gamma-phosphate of ATP in the transition state, and this is likely to be a general feature of aminoacyl-tRNA synthetases that have a HIGH region.
Department of Chemistry, Imperial College of Science and Technology, London, U.K.
T J
TJ
Borgford
T E
TE
Gray
N J
NJ
Brand
A R
AR
Fersht
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D005796:Genes; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D009154:Mutation; D013816:Thermodynamics; D014637:Valine-tRNA Ligase / Q000235:genetics*
D000604:Amino Acyl-tRNA Synthetases; D014637:Valine-tRNA Ligase