Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action
Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action
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DOI：
10.1096/fj.11-191254
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发表时间：
2012-03-01
期刊：
影响因子：
4.8
通讯作者：
Kundu, Bishwajit
中科院分区：
文献类型：
--
作者：
Bansal, Saurabh;Srivastava, Ankit;Kundu, Bishwajit
嗜热l -天冬酰胺酶在高温下表现出很高的稳定性和活性。然而，它们在白血病治疗中的应用有限，因为它们的底物亲和力低，在生理条件下活性降低。为了将稳定性与生理条件下的活性结合起来，开发了3个荧光焦球菌l -天冬酰胺酶（PfA）的活性位点突变体。突变体，特别是K274E，在生理条件下表现出比野生型更好的酶特性。所有的变异都是热力学稳定的，并且抵抗蛋白水解消化。没有一种酶显示谷氨酰胺酶活性，这是一种非常理想的治疗特性。与大肠杆菌l -天冬酰胺酶相比，所有变体对人细胞株HL60、MCF7和K562的杀伤作用都更高、更显著。我们的研究表明，通过活性位点环增加的底物可及性在决定活性方面起主要作用。基于分子动力学模拟结构，提出了一种新的机制见解，其中关键Tyr残基的动态翻转是嗜热l -天冬酰胺酶活性的原因。我们的研究不仅开发了具有理想特性的PfA突变体，而且还提供了有关其活性的机制见解。-Bansal， S., Srivastava， A., Mukherjee， G., Pandey， R., Verma， A. K., Mishra， P., Kundu， B.具有增强底物亲和力和抗肿瘤活性的超嗜热天冬酰胺酶突变体：对其作用机制的结构见解。中国生物医学工程学报，26(4):559 - 561（2012）。www.fasebj.org
Thermophilic L-asparaginases display high stability and activity at elevated temperatures. However, they are of limited use in leukemia therapy because of their low substrate affinity and reduced activity under physiological conditions. In an attempt to combine stability with activity at physiological conditions, 3 active-site mutants of Pyrococcus furiosus L-asparaginase (PfA) were developed. The mutants, specifically K274E, showed improved enzymatic properties at physiological conditions as compared to the wild type. All variants were thermodynamically stable and resistant to proteolytic digestion. None of the enzymes displayed glutaminase activity, a highly desirable therapeutic property. All variants showed higher and significant killing of human cell lines HL60, MCF7, and K562 as compared to the Escherichia coli L-asparaginase. Our study revealed that increased substrate accessibility through the active site loop plays a major role in determining activity. A new mechanistic insight has been proposed based on molecular dynamics simulated structures, where dynamic flipping of a critical Tyr residue is responsible for the activity of thermophilic L-asparaginases. Our study not only resulted in development of PfA mutants with combination of desirable properties but also gave a mechanistic insight about their activity.-Bansal, S., Srivastava, A., Mukherjee, G., Pandey, R., Verma, A. K., Mishra, P., Kundu, B. Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action. FASEB J. 26, 1161-1171 (2012). www.fasebj.org