Brown K A, Brick P, Blow D M
Nature. 1987;326(6111):416-8. doi: 10.1038/326416a0.
One surprising outcome of applying the techniques of protein engineering to the enzyme tyrosyl-transfer RNA synthetase was that the enzyme's activity could actually be increased by a specific sequence change. In particular, altering residue threonine 51 to a proline (mutant TP51) increased the enzyme's affinity for tyrosyl adenylate complexes. The non-additive effect of combining the TP51 mutation with a second sequence alteration (histidine 48 to a glycine) suggested that the effect of the TP51 change might be mediated by a structural change involving the peptide backbone. To address the question of the mechanism by which the TP51 change increases the activity of tyrosyl-tRNA synthetase we have determined the structure of the mutant enzyme. We find the change has a purely local effect on the structure of the enzyme, and conclude that the increased activity of the TP51 mutant probably results from the replacement of the polar threonine residue by a non-polar group: in the wild-type enzyme substrate binding is disfavoured by the displacement of solvent from the vicinity of threonine 51. This unfavourable effect is absent in the TP51 mutant.
将蛋白质工程技术应用于酪氨酰 - 转移RNA合成酶时，一个令人惊讶的结果是，通过特定的序列改变实际上可以提高该酶的活性。具体而言，将苏氨酸51残基改变为脯氨酸（突变体TP51）增加了该酶对酪氨酰腺苷酸复合物的亲和力。将TP51突变与第二个序列改变（组氨酸48变为甘氨酸）相结合的非加性效应表明，TP51改变的效应可能是由涉及肽主链的结构变化介导的。为了解决TP51改变增加酪氨酰 - tRNA合成酶活性的机制问题，我们测定了突变酶的结构。我们发现这种改变对酶的结构具有纯粹的局部影响，并得出结论，TP51突变体活性增加可能是由于极性苏氨酸残基被非极性基团取代所致：在野生型酶中，底物结合因苏氨酸51附近溶剂的置换而不利。在TP51突变体中不存在这种不利影响。
