通过 2D-ITC 以高分辨率表征双底物酶动力学,Analytical Chemistry

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通过 2D-ITC 以高分辨率表征双底物酶动力学
Analytical Chemistry ( IF 7.4 ) Pub Date : 2021-09-12 , DOI: 10.1021/acs.analchem.1c02705
Yun Wang ₁ , Anthony K Mittermaier ₁

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人们越来越关注使用等温滴定量热法 (ITC) 通过实时测量催化产生或吸收的热量来表征酶动力学。由于几乎所有的化学反应都与焓的变化有关，因此 ITC 代表了一种稳健且几乎通用的实验方法。尽管如此，仍有一些技术挑战限制了 ITC 的适用性。例如，含有两种底物的酶（双底物酶）的完整动力学表征，包括大多数已知例子，需要进行一系列实验，因为两种底物的浓度是变化的。这是使用当前 ITC 方法的耗时且昂贵的过程，因为必须独立执行许多 (> 5) 个单独的实验以获得足够数量的数据。我们开发了一种新的 ITC 方法，我们将其称为 2D-ITC，该方法在一次大约 2 小时长的实验中将反应速度映射为两种底物浓度的函数。这种方法提供的细节水平可与任何现有的酶测定相媲美或超过任何现有的酶测定法，因为单个实验产生了 7000 次催化速率测量。在对兔肌肉丙酮酸激酶 (rMPK) 的原理验证应用中，该方法正确识别了该酶的随机序列机制和氨基酸苯丙氨酸 (Phe) 的变构催化抑制。出乎意料的是，我们发现虽然 Phe 降低了对底物磷酸烯醇丙酮酸的亲和力，这是一种已知的现象，但它也减轻了反应产物 ATP 的抑制作用，这在以前没有报道过。鉴于细胞中 ATP 的相对丰度，

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Characterizing Bi-substrate Enzyme Kinetics at High Resolution by 2D-ITC

There is growing interest in using isothermal titration calorimetry (ITC) to characterize enzyme kinetics by measuring the heat produced or absorbed by catalysis in real time. Since virtually all chemical reactions are associated with changes in enthalpy, ITC represents a robust and nearly universal experimental approach. Nevertheless, there are technical challenges that limit ITC’s applicability. For instance, the full kinetic characterization of enzymes with two substrates (bi-substrate enzymes), which comprise the majority of known examples, requires a series of experiments to be performed as the concentrations of both substrates are varied. This is a time-consuming and expensive process using current ITC methods since many (>5) individual experiments must be performed independently to obtain a sufficient quantity of data. We have developed a new ITC method, which we term 2D-ITC, which maps the reaction velocity as a function of two substrate concentrations in a single, roughly 2 h long experiment. This method provides a level of detail that rivals or exceeds any existing enzyme assay, as a single experiment generates on the order of 7000 catalytic rate measurements. In a proof-of-principle application to rabbit muscle pyruvate kinase (rMPK), the method correctly identified the enzyme’s random sequential mechanism and allosteric catalytic suppression by the amino acid phenylalanine (Phe). Unexpectedly, we found that while Phe reduces affinity for the substrate phosphoenolpyruvate, a known phenomenon, it also alleviates inhibition by the reaction product ATP, which had not been reported previously. Given the relative abundance of ATP in the cell, this opposing effect is expected to have a substantial impact on rMPK activity.

更新日期：2021-09-21

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