Biophysics & Structural Genomics, Saha Institute of Nuclear Physics, Kolkata 700064, India.
Homi Bhabha National Institute, Anushaktinagar, Mumbai 400094, India.
J Am Chem Soc. 2023 May 17;145(19):10826-10838. doi: 10.1021/jacs.3c02154. Epub 2023 May 8.
l-asparaginase II (MW 135 kDa) from is an FDA-approved protein drug used for the treatment of childhood leukemia. Despite its long history as a chemotherapeutic, the structural basis of enzyme action, in solution, remains widely contested. In this work, methyl-based 2D [H-C]-heteronuclear single-quantum correlation (HSQC) NMR, at natural abundance, has been used to profile the enzymatic activity of the commercially available enzyme drug. The [H-C]-HSQC NMR spectra of the protein reveal the role of a flexible loop segment in the activity of the enzyme, in solution. Addition of asparagine to the protein results in distinct conformational changes of the loop that could be signatures of intermediates formed in the catalytic reaction. To this end, an isothermal titration calorimetry (ITC)-based assay has been developed to measure the enzymatic reaction enthalpy, as a marker for its activity. Combining both ITC and NMR, it was shown that the disruption of the protein conformation can result in the loss of function. The scope, robustness, and validity of the loop fingerprints in relation to enzyme activity have been tested under different solution conditions. Overall, our results indicate that 2D NMR can be used reliably to gauge the structure-function of this enzyme, bypassing the need to label the protein. Such natural abundant NMR methods can be potentially extended to probe the structure-function aspects of high-molecular-weight protein therapeutics (glycosylated protein drugs, enzymes, therapeutic monoclonal antibodies, antibody-drug conjugates, and Fc-fusion proteins), where (a) flexible loops are required for their function and (b) isotope labeling may not be straightforward.
来自 的天冬酰胺酶 II（MW135kDa）是一种经美国食品和药物管理局批准的用于治疗儿童白血病的蛋白药物。尽管它作为化疗药物已有很长的历史，但酶在溶液中的作用的结构基础仍存在广泛争议。在这项工作中，采用基于氘甲基的二维 [H-C]-异核单量子相关（HSQC）NMR（在自然丰度下）来分析市售酶药物的酶活性。该蛋白的 [H-C]-HSQC NMR 谱揭示了在溶液中，一个灵活的环段在酶活性中的作用。向蛋白质中添加天冬酰胺会导致环的构象发生明显变化，这可能是催化反应中形成的中间产物的特征。为此，开发了一种基于等温热力学滴定（ITC）的测定法来测量酶反应焓，作为其活性的指标。通过结合 ITC 和 NMR，结果表明蛋白质构象的破坏会导致功能丧失。在不同的溶液条件下，已经测试了环指纹与酶活性的相关性的范围、稳健性和有效性。总体而言，我们的结果表明，二维 NMR 可用于可靠地评估该酶的结构-功能，而无需对蛋白质进行标记。这种天然丰度的 NMR 方法可潜在地扩展到研究高分子量蛋白治疗剂（糖基化蛋白药物、酶、治疗性单克隆抗体、抗体药物偶联物和 Fc 融合蛋白）的结构-功能方面，其中（a）灵活的环对于它们的功能是必需的，和（b）同位素标记可能并不简单。
