Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-asparaginase
Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-asparaginase
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DOI：
10.1021/bi0029595
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发表时间：
2001-05-15
期刊：
影响因子：
2.9
通讯作者：
Lubkowski, J
中科院分区：
文献类型：
--
作者：
Aghaiypour, K;Wlodawer, A;Lubkowski, J
细菌L-天冬酰胺酶，催化L-天冬酰胺水解为天冬氨酸的酶，已被用作治疗急性儿童淋巴细胞白血病的治疗剂超过30年。天冬酰胺酶的其他底物包括L-谷氨酰胺、D-天冬酰胺。和琥珀酸单酰胺。在这份报告中，我们提出了高分辨率的晶体结构的复合物欧文氏菌的L-天冬酰胺酶（ErA）与这种反应的产物，也可以作为底物，即L-谷氨酸（L-Glu），D-天冬氨酸（D-Asp），和琥珀酸（Suc）。每个复合物内的四个独立活性位点的比较表明配体分子的独特和特异性结合;复合物之间的结合模式也相似。与L-Asp相比，Sue中缺乏α-NH3+基团不影响结合模式。L-Glu的侧链大于L-Asp的侧链，导致ErA活性侧的几种结构扭曲。活性位点柔性环（残基15-33）不表现出稳定的构象，导致亲核试剂Thr 15的次优取向。此外，L-Glu的δ-COO-平面近似垂直于与天冬酰胺酶活性位点结合的L-Asp中的γ-COO-平面。与其它配体相比，D-Asp与ErA活性位点的结合是非常独特的，这表明ErA对D-Asp的低活性可以主要归因于低k（cat）值。ErA与其他细菌L-天冬酰胺酶的氨基酸序列和晶体结构的比较表明，两个活性位点残基Glu 63（ErA）和Ser 254（ErA）的存在可能与显著的天冬酰胺酶活性相关，而它们分别被Gln和Asn取代可能导致最小的L-天冬酰胺酶活性。
Bacterial L-asparaginases, enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid, have been used for over 30 years as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Other substrates of asparaginases include L-glutamine, D-asparagine. and succinic acid monoamide. In this report, we present high-resolution crystal structures of the complexes of Erwinia chrysanthemi L-asparaginase (ErA) with the products of such reactions that also can serve as substrates, namely L-glutamic acid (L-Glu), D-aspartic acid (D-Asp), and succinic acid (Suc). Comparison of the four independent active sites within each complex indicates unique and specific binding of the ligand molecules; the mode of binding is also similar between complexes. The lack of the alpha -NH3+ group in Sue, compared to L-Asp, does not affect the binding mode. The side chain of L-Glu, larger than chat of L-Asp, causes several structural distortions in the ErA active side. The active site flexible loop (residues 15-33) does not exhibit stable conformation, resulting in suboptimal orientation of the nucleophile, Thr15. Additionally, the delta -COO- plane of L-Glu is approximately perpendicular to the plane of gamma -COO- in L-Asp bound to the asparaginase active site. Binding of D-Asp to the ErA active site is very distinctive compared to the other ligands, suggesting that the low activity of ErA against D-Asp could be mainly attributed to the low k(cat) value. A comparison of the amino acid sequence and the crystal structure of ErA with those of other bacterial L-asparaginases shows that the presence of two active-site residues, Glu63(ErA) and Ser254(ErA), may correlate with significant glutaminase activity, while their substitution by Gin and Asn, respectively, may lead to minimal L-glutaminase activity.