Abstract
A single point mutation has been engineered in the tyrosyl-tRNA synthetase that improves its affinity (KM) for its substrate ATP by a factor of 100. In the crystal structure of the tyrosyl tRNA synthetase (of Bacillus stearothermophilus), the side-chain hydroxyl of Thr 51 appears to make a weak hydrogen bond with the AMP moiety of the substrate intermediate, tyrosyl adenylate. In the absence of substrate, however, the hydroxyl group should make a strong hydrogen bond with water which would favour dissociation of the enzyme–substrate complex. We have used oligodeoxynucleotide-directed mutagenesis to construct two point mutants at this site: one to remove the hydroxyl group (Thr 51 → Ala 51) and the other, in addition, to distort the local polypeptide backbone (Thr 51 → Pro 51). We report here that both mutants have increased activity (kcat/KM for ATP) but one mutant (Pro 51) shows a massive 25-fold increase due mainly to a lowered KM for ATP. This demonstrates dramatically the potential of in vitro mutagenesis for improving the affinity of an enzyme for its substrate.
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Wilkinson, A., Fersht, A., Blow, D. et al. A large increase in enzyme–substrate affinity by protein engineering. Nature 307, 187–188 (1984). https://doi.org/10.1038/307187a0
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DOI: https://doi.org/10.1038/307187a0
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