Crystal structure of adenosine 5′-phosphosulfate kinase from Penicillium chrysogenum
Crystal structure of adenosine 5′-phosphosulfate kinase from Penicillium chrysogenum
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DOI：
10.1021/bi9924157
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发表时间：
2000-02-22
期刊：
影响因子：
2.9
通讯作者：
Fisher, AJ
中科院分区：
文献类型：
--
作者：
MacRae, IJ;Segel, IH;Fisher, AJ
腺苷5 '-磷酸硫酸盐（APS）激酶催化无机硫酸盐两步转化为3'-磷酸腺苷5 '-磷酸硫酸盐（PAPS）的第二反应。本文报道了丝状真菌产黄青霉无配体APS激酶的2.0埃分辨率晶体结构。该酶结晶为同源二聚体，每个亚基折叠成经典的激酶基序，由夹在两个或-螺旋束之间的扭曲的平行β-片层组成。步行者A基序（32）GLSASGKS（39）形成了预测的P环结构。叠加的APS激酶活性位点区域到其他几个P-环的蛋白质显示，保守的天冬氨酸残基，通常相互作用的Mg 2+的MgATP的协调领域是不存在的APS激酶。然而，MgATP结合后，不同的天冬氨酸，天冬氨酸61，可以移动和结合的Mg 2+。序列（156）KAREGVIKEFT（166）被认为是（P）APS基序，位于高度蛋白酶敏感的环中，该环在游离酶的两个亚基中都是无序的。MgATP或MgADP可防止蛋白水解;单独使用APS没有效果，但可增强MgADP提供的保护。结果表明，在MgATP或MgADP结合之前，该环缺乏固定结构。随后的构象变化以及由MgATP与Asp 61相互作用促进的潜在变化可以定义APS结合位点。该模型与根据稳态动力学和平衡结合研究建立的强制性有序底物结合序列（MgATP或APS之前的MgADP）一致。
Adenosine 5'-phosphosulfate (APS) kinase catalyzes the second reaction in the two-step conversion of inorganic sulfate to 3'-phosphoadenosine 5'-phosphosulfate (PAPS). This report presents the 2.0 Angstrom resolution crystal structure of ligand-free APS kinase from the filamentous fungus, Penicillium chrysogenum. The enzyme crystallized as a homodimer with each subunit folded into a classic kinase motif consisting of a twisted, parallel beta-sheet sandwiched between two or-helical bundles. The Walker A motif, (32)GLSASGKS(39), formed the predicted P-loop structure. Superposition of the APS kinase active site region onto several other P-loop-containing proteins revealed that the conserved aspartate residue that usually interacts with the Mg2+ coordination sphere of MgATP is absent in APS kinase. However, upon MgATP binding, a different aspartate, Asp 61, could shift and bind to the Mg2+. The sequence (156)KAREGVIKEFT(166), which has been suggested to be a (P)APS motif, is located in a highly protease-susceptible loop that is disordered in both subunits of the free enzyme. MgATP or MgADP protects against proteolysis; APS alone has no effect but augments the protection provided by MgADP. The results suggest that the loop lacks a fixed structure until MgATP or MgADP is bound. The subsequent conformational change together with the potential change promoted by the interaction of MgATP with Asp 61 may define the APS binding site. This model is consistent with the obligatory ordered substrate binding sequence (MgATP or MgADP before APS) as established from steady state kinetics and equilibrium binding studies.