Comparison of rates and kinetic isotope effects using PEG-modified variants and glycoforms of glucose oxidase: the relationship of modification of the protein envelope to C-H activation and tunneling.
Comparison of rates and kinetic isotope effects using PEG-modified variants and glycoforms of glucose oxidase: the relationship of modification of the protein envelope to C-H activation and tunneling.
复制标题
使用葡萄糖氧化酶的 PEG 修饰变体和糖型进行速率和动力学同位素效应的比较：蛋白质包膜修饰与 C-H 激活和隧道效应的关系。
DOI：
10.1021/bi020054g
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Klinman,JudithP
中科院分区：
文献类型：
--
作者：
Seymour,SeanL;Klinman,JudithP
早期对葡萄糖氧化酶 (GO) 中速率和动力学同位素效应 (KIE) 的温度依赖性的研究使用了蛋白质表面糖基化程度不同的变体。科恩等人。 [Kohen, A., Jonsson, T., 和 Klinman, J. P. (1997)Biochemistry36, 2603−2611] 提出了阿伦尼乌斯前因子上的 KIE 作为蛋白质修饰的函数而变化的证据，得出的结论是，活性位点的氢隧道程度取决于表面质量的变化。我们现在检查表面含有聚乙二醇 (PEG) 的 GO 蛋白和更广泛的糖基化形式的 GO，以区分简单的质量效应和其他催化行为改变的来源。一种 PEG 变体是通过用短 PEG 链（平均每条 350 Da）修饰去糖基化 GO 来创建的，而另一种则包含少量的长 PEG 链（平均每条 5000 Da）。轻 (146 kDa) 和重 (211 kDa) PEG 变体以及高糖基化变体对阿伦尼乌斯前因子表现出相似的同位素效应 (AD/AT= 0.55−0.62)，而未受干扰的野生型 GO (WT-GO) 被发现具有 AD/AT，经重新评估，该 AD/AT 接近统一。似乎对蛋白质表面进行任何远离野生型表面的修饰都会导致活性位点氢转移行为的改变。我们还比较了 kcat/KM 和 kcat 变体的活化焓的影响，引入了一种从竞争性 KIE 实验中提取氚化底物的 kcat/KMrate 常数和活化焓的新方法。我们发现竞争性和非竞争性参数的 ΔH⧧ 趋势相似，并且 inkcat 的趋势比之前报告的更小。观察到 AD/AT 与 GO 异构体的活化焓和热熔化温度 (TM) 之间存在相关性。除了本研究之外，现在还有许多例子，其中酶结构的扰动远离野生型，导致观察到的 KIE 变得更加依赖于温度。这些发现的含义在氢隧道以及蛋白质结构和动力学与该过程的关系的背景下进行了讨论。
An earlier investigation of the temperature dependencies of rates and kinetic isotope effects (KIEs) in glucose oxidase (GO) used variants that differed in the extent of glycosylation at the surface of the protein. Kohen et al. [Kohen, A., Jonsson, T., and Klinman, J. P. (1997)Biochemistry36, 2603−2611] presented evidence that the KIE on the Arrhenius prefactor varied as a function of protein modification, concluding that the degree of hydrogen tunneling at the active site was dependent on changes in mass at the surface. We now examine GO proteins containing polyethylene glycol (PEG) at their surface and a more extensively glycosylated form of GO, to distinguish simple mass effects from other sources of altered catalytic behavior. One PEG variant was created by modifying deglycosylated GO with short PEG chains (average of 350 Da each), while another contained a smaller number of long PEG chains (average of 5000 Da each). The light (146 kDa) and heavy (211 kDa) PEG variants and the hyperglycosylated variant display isotope effects on the Arrhenius prefactor that are similar (AD/AT= 0.55−0.62), while the unperturbed wild-type GO (WT-GO) is found to have anAD/ATthat is reassessed as being close to unity. It appears that any modification of the protein surface away from that of the wild type gives rise to altered behavior for hydrogen transfer in the active site. We have also compared the effect of enthalpies of activation on bothkcat/KMandkcatfor the variants, introducing a new method to extract thekcat/KMrate constant and enthalpy of activation for the tritiated substrate from competitive KIE experiments. We find similar trends in ΔH⧧for both competitive and noncompetitive parameters and a smaller trend inkcatthan reported earlier. Correlations are observed betweenAD/ATand both the enthalpies of activation and the thermal melt temperatures (TM) of the GO isoforms. In addition to the present study, there are now a number of examples where a perturbation of enzyme structure away from that of the wild type causes the observed KIE to become more temperature-dependent. The implications of these findings are discussed in the context of hydrogen tunneling and the relationship of protein structure and dynamics to this process.