Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus
Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus
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DOI：
10.1134/s0006297910030144
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发表时间：
2010-03-01
影响因子：
2.8
通讯作者：
Kundu, B.
中科院分区：
文献类型：
--
作者：
Bansal, S.;Gnaneswari, D.;Kundu, B.
我们报道了一个L-天冬酰胺酶的研究，从激烈的火球菌，克隆和表达在大肠杆菌和纯化到同质。蛋白质的稳定性和酶动力学参数进行了测定。该酶被认为是热稳定的，天然二聚体，和精氨酸酶，在pH 9.0的最佳活性。其显示12 mM的K（m）和高于20 mM L-天冬酰胺的底物抑制曲线。尿素不能诱导展开和酶失活，然而，与盐酸胍（GdnCl）的两个状态展开模式进行了观察。在低GdnCl浓度（1 M）下蛋白质的活性降低和近UV-CD信号改变表明酶活性位点处的三级结构变化。同源三维模型的开发和结构信息相结合的活性和稳定性数据，提供功能线索的天冬酰胺酶。
We report studies on an L-asparaginase from Pyrococcus furiosus, cloned and expressed in Escherichia coli and purified to homogeneity. Protein stability and enzyme kinetic parameters were determined. The enzyme was found to be thermostable, natively dimeric, and glutaminase-free, with optimum activity at pH 9.0. It showed a K (m) of 12 mM and a substrate inhibition profile above 20 mM L-asparagine. Urea could not induce unfolding and enzyme inactivation; however, with guanidine hydrochloride (GdnCl) a two-state unfolding pattern was observed. Reduced activity and an altered near-UV-CD signal for protein at low GdnCl concentration (1 M) suggested tertiary structural changes at the enzyme active site. A homology three-dimensional model was developed and the structural information was combined with activity and stability data to give functional clues about the asparaginase.