TRANSITION-STATE STABILIZATION IN THE MECHANISM OF TYROSYL-TRANSFER RNA-SYNTHETASE REVEALED BY PROTEIN ENGINEERING
TRANSITION-STATE STABILIZATION IN THE MECHANISM OF TYROSYL-TRANSFER RNA-SYNTHETASE REVEALED BY PROTEIN ENGINEERING
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DOI：
10.1073/pnas.82.23.7840
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
WINTER, G
中科院分区：
文献类型：
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作者：
LEATHERBARROW, RJ;FERSHT, AR;WINTER, G
研究发现，酪氨酰-tRNA 合成酶激活酪氨酸的主要催化因素是过渡态 ATP 结合的改善。活化反应涉及酪氨酰羧酸盐对ATP的α-磷酸基团的攻击以产生五配位过渡态。该复合物的模型构建定位了 ATP γ-磷酸基团的结合位点，该结合位点由与 Thr-40 和 His-45 侧链的氢键组成。通过蛋白质工程去除这些基团表明它们不贡献与未反应的 ATP 的结合能，而是将所有结合能用于稳定 [酪氨酸 .cntdot。 ATP]过渡态[突变酪氨酰-tRNA合成酶(Thr-40.fwdarw.Ala-40；His-45.fwdarw.Gly-45)酪氨酰腺苷酸的形成速率降低3.2倍。 105，但 ATP 的 KS 仅降低了 5 倍]。这些残基的侧链还在逆反应中提供焦磷酸的结合位点。因此，催化作用伴随着通过改善远离反应位点的底物上的基团的结合来稳定过渡态。
The principal catalytic factor in the activation of tyrosine by the tyrosyl-tRNA synthetase is found to be improved binding of ATP in the transition state. The activation reaction involves the attack of the tyrosyl carboxylate on the .alpha.-phosphate group of ATP to generate a pentacoordinate transition state. Model building of this complex located a binding site for the .gamma.-phosphate group of ATP, consisting of hydrogen bonds with the side chains of Thr-40 and His-45. Removal of these groups by protein engineering shows that they contribute no binding energy with unreacted ATP but put all of their binding energy into stabilizing the [tyrosine .cntdot. ATP] transition state [the mutant tyrosoyl-tRNA synthetase(Thr-40 .fwdarw. Ala-40; His-45 .fwdarw. Gly-45) has the rate of formation of tyrosyl adenylate lowered by 3.2 .times. 105 but KS for ATP is lowered by only a factor of 5]. The side chains of these residues also provide a binding site for pyrophosphate in the reverse reaction. Thus, catalysis is accompanied by stabilization of the transition state by improved binding of a group on the substrate that is distant from the seat of reaction.