QUANTITATIVE INTERPRETATIONS OF DOUBLE MUTATIONS OF ENZYMES
QUANTITATIVE INTERPRETATIONS OF DOUBLE MUTATIONS OF ENZYMES
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DOI：
10.1016/0003-9861(92)90692-p
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发表时间：
1992-05-01
影响因子：
3.9
通讯作者：
KULIOPULOS, A
中科院分区：
文献类型：
--
作者：
MILDVAN, AS;WEBER, DJ;KULIOPULOS, A
第二突变对突变酶的定量效应相对于第一突变可以是拮抗的、不存在的、部分加和的、加和的或协同的。根据测定的酶的动力学或热力学参数，相同的两个突变在双突变体中可以不同地相互作用。两个突变对平衡常数的加和效应，例如酶-底物复合物的解离常数（Ks），当促进相同步骤的非相互作用残基（底物结合）发生突变。部分累加效应是由突变的两个残基与底物的协同相互作用引起的，协同效应是由突变的两个残基与底物的反协同作用引起的。协同作用的另一种解释是酶的广泛解折叠。对平衡常数如K s的拮抗作用是由两个突变对底物结合的相反结构效应引起的。在双突变体中第二个突变没有额外的作用代表了部分加和或协同作用的限制性情况。两个突变对速率常数的影响的相互作用，如kcathave与上面对平衡常数给出的解释相同，因为发生了限速过渡态的结合。然而，由于动力学的复杂性，存在以下例外和补充。当促进相同步骤的非相互作用残基发生突变时，两个突变对kcat的累加效应发生，前提是该步骤是速率限制的。如果受影响的步骤不是速率限制的，则观察到两个突变的协同效应，因为每个突变导致步骤逐渐变得更加速率限制。当两个突变影响连续的步骤时，也会发生对kcat的加和效应，只要其中一个是速率限制的。当促进连续的、非限速步骤的非相互作用残基发生突变时，也会发生对kcat的部分加性效应。这些概念，当应用于已发表的Δ5-3-酮类固醇异构酶，葡萄球菌核酸酶，酪氨酰-tRNA合成酶，谷胱甘肽还原酶和枯草杆菌蛋白酶的双突变体的数据时，提供了对氨基酸残基在结合底物，激活剂和抑制剂以及促进催化中的独立，合作，反合作或拮抗相互作用的更深入的见解。
The quantitative effect of a second mutation on a mutant enzyme may be antagonistic, absent, partially additive, additive, or synergistic with respect to the first mutation. Depending on which kinetic or thermodynamic parameter of an enzyme is measured, the same two mutations can interact differently in the double mutant.Additive effectsof two mutations on an equilibrium constant, such as the dissociation constant of the enzyme-substrate complex (Ks), occur when noninteracting residues which facilitate the same step (substrate binding) are mutated.Partially additive effectsresult from the cooperative interaction with the substrate of the two residues mutated, andsynergistic effectsresult from the anticooperative interaction with the substrate of the two residues mutated. An alternative explanation for synergy is extensive unfolding of the enzyme.Antagonisticeffects on an equilibrium constant such asKsresult from opposing structural effects of the two mutations on substrate binding.No additional effectof the second mutation in the double mutant represents a limiting case of eitherpartial additivityorsynergy. The interactions of the effects of two mutations on a rate constant such askcathave the same explanations as those given above for equilibrium constants since the binding of a rate-limiting transition state is occurring. However, due to kinetic complexity, the following exceptions and additions exist. Additive effects of two mutations onkcatoccur when non-interacting residues which facilitate the same step are mutated, provided this step is rate limiting. If the affected step is not rate limiting thensynergisticeffects of the two mutations are observed as each mutation causes the step to become progressively more rate limiting. Additive effects onkcatalso occur when the two mutations affect consecutive steps, provided one of them is rate limiting. Partially additive effects onkcatalso occur when noninteracting residues facilitating consecutive, non-rate-limiting steps are mutated. These concepts, when applied to published data on double mutants of Δ5-3-ketosteroid isomerase, staphylococcal nuclease, tyrosyl-tRNA synthetase, glutathione reductase, and subtilisin, provide deeper insights into the independent, cooperative, anti-cooperative, or antagonistic interactions of amino acid residues in the binding of substrates, activators, and inhibitors and in promoting catalysis.