Temperature-dependent isotope effects in soybean lipoxygenase-1: Correlating hydrogen tunneling with protein dynamics
Temperature-dependent isotope effects in soybean lipoxygenase-1: Correlating hydrogen tunneling with protein dynamics
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DOI：
10.1021/ja012205t
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发表时间：
2002-04-17
影响因子：
15
通讯作者：
Klinman, JP
中科院分区：
文献类型：
--
作者：
Knapp, MJ;Rickert, K;Klinman, JP
大豆脂氧合酶-1（SLO）的氢原子转移在室温附近表现出很大的动力学同位素效应（KIE = 81）和很弱的温度依赖性（E-act = 2.1kcal/mol）。这些性质与H.完全由隧道事件发生的传输。制备SLO的突变体，并测量KIE的温度依赖性，以测试隧穿行为的改变。研究的所有突变体在30 ℃下表现出相似的大幅度KIES，尽管k（cat）的变化高达3个数量级。两个突变体（Leu（754）--> Ala，Leu（546）--> Ala）的E-act大于野生型（WT），并且KIE变得稍微更依赖于温度。相反，Ile（553）-> Ala对质子化底物表现出与野生型大豆脂氧合酶-1（WT-SLO）相似的k（cat）和E-act参数;然而，KIE显著地依赖于温度。前两个突变体的行为可以反映增加的重组能（λ），但后一个突变体的行为与此描述不一致。我们已经全面启动了。隧道模型（Kuznetsov，A. M.;乌尔斯特鲁普，J. Can. 1999，77，1085-1096）来解释KIE的温度依赖性，其指示距离取样（门控）调节氢转移的程度。WT-SLO表现出非常小的E-act和几乎与温度无关的KIE，其被建模为由压缩的氢转移距离产生，几乎没有氢转移距离的调制。对Leu（754）-> Ala和Leu（546）-> Ala突变体的观察被建模为产生于稍微较少压缩的活性位点，其具有由环境动力学对氢转移距离的较大调节。最后，观察到的Ile（553）-> Ala突变体的行为表明松弛的活性位点广泛参与门控以促进氢转移。我们的结论是，WT-SLO有一个活跃的网站结构，组织良好，以支持氢隧道和突变扰动结构元素，支持氢隧道。活性位点残基的适度改变增加λ和/或增加氢转移距离，从而影响隧穿可能发生的概率。这些研究允许检测和表征催化中的蛋白质门控模式。
The hydrogen-atom transfer in soybean lipoxygenase-1 (SLO) exhibits a large kinetic isotope effect on k(cat) (KIE = 81) near room temperature and a very weak temperature dependence (E-act = 2.1 kcal/mol). These properties are consistent with H. transfer that occurs entirely by a tunneling event. Mutants of SLO were prepared, and the temperature dependence of the KIE was measured, to test for alterations in the tunneling behavior. All mutants studied exhibit KIES of similar, large magnitude at 30 degreesC, despite an up to 3 orders of magnitude change in k(cat). E-act for two of the mutants (Leu(754) --> Ala, Leu(546) --> Ala) is larger than for wild-type (WT), and the KIE becomes slightly more temperature dependent. In contrast, Ile(553) --> Ala exhibits k(cat) and E-act parameters similar to wild-type soybean lipoxygenase-1 (WT-SLO) for protiated substrate; however, the KIE is markedly temperature dependent. The behavior of the former two mutants could reflect increased reorganization energies (lambda), but the behavior of the latter mutant is inconsistent with this description. We have invoked a full H-. tunneling model (Kuznetsov, A. M.; Ulstrup, J. Can. J. Chem. 1999, 77, 1085-1096) to explain the temperature dependence of the KIE, which is indicative of the extent to which distance sampling (gating) modulates hydrogen transfer. WT-SLO exhibits a very small E-act and a nearly temperature-independent KIE, which was modeled as arising from a compressed hydrogen transfer distance with little modulation of the hydrogen transfer distance. The observations on the Leu(754) --> Ala and Leu(546) --> Ala mutants were modeled as arising from a slightly less compressed active site with greater modulation of the hydrogen transfer distance by environmental dynamics. Finally, the observed behavior of the Ile(553) --> Ala mutant indicates a relaxed active site with extensive involvement of gating to facilitate hydrogen transfer. We conclude that WT-SLO has an active site structure that is well organized to support hydrogen tunneling and that mutations perturb structural elements that support hydrogen tunneling. Modest alterations in active site residues increase lambda and/or increase the hydrogen transfer distance, thereby affecting the probability that tunneling can occur. These studies allow the detection and characterization of a protein-gating mode in catalysis.