自 20 世纪 70 年代以来，来自大肠杆菌 (EcA) 的 L-天冬酰胺酶一直用于治疗急性淋巴细胞白血病 (ALL)。然而，这种酶具有第二种特异性，会导致谷氨酰胺酶分解，导致患者体内消耗，从而造成严重的副作用。尽管人们对这种酶的使用产生了巨大的兴趣，但谷氨酰胺消耗的确切过程仍然未知，并且对于 L-天冬酰胺水解也没有达成共识。在这里，我们研究了 T12、Y25 和 T89 在天冬酰胺酶和谷氨酰胺酶活性中的作用。我们获得了含有 T12、Y25 或 T89 残基突变的单个克隆。重组生产野生型和突变型 EcA 后，使用纳米差示扫描荧光法对纯化的样品进行结构分析，结果表明所有样品均以其活性结构构象（同四聚体构象）含有热稳定性分子。四级构象经DLS和SEC确认。活性酶测定结合分子动力学模拟确定了 T12、Y25 和 T89 残基对 EcA 谷氨酰胺酶和天冬酰胺酶活性的贡献。我们的结果绘制了酶行为图，为设计改进的 EcA 酶铺平了道路，这对于治疗 ALL 非常重要。版权所有 © 2023。由 Elsevier B.V 出版。

L-asparaginase from Escherichia coli (EcA) has been used for the treatment of acute lymphoid leukemia (ALL) since the 1970s. Nevertheless, the enzyme has a second specificity that results in glutaminase breakdown, resulting in depletion from the patient's body, causing severe adverse effects. Despite the huge interest in the use of this enzyme, the exact process of glutamine depletion is still unknown and there is no consensus regarding L-asparagine hydrolysis. Here, we investigate the role of T12, Y25, and T89 in asparaginase and glutaminase activities. We obtained individual clones containing mutations in the T12, Y25 or T89 residues. After the recombinant production of wild-type and mutated EcA, The purified samples were subjected to structural analysis using Nano Differential Scanning Fluorimetry, which revealed that all samples contained thermostable molecules in their active structural conformation, the homotetramer conformation. The quaternary conformation was confirmed by DLS and SEC. The activity enzymatic assay combined with molecular dynamics simulation identified the contribution of T12, Y25, and T89 residues in EcA glutaminase and asparaginase activities. Our results mapped the enzymatic behavior paving the way for the designing of improved EcA enzymes, which is important in the treatment of ALL.Copyright © 2023. Published by Elsevier B.V.