Reconstituted IMPDH polymers accommodate both catalytically active and inactive conformations.
Reconstituted IMPDH polymers accommodate both catalytically active and inactive conformations.
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DOI：
10.1091/mbc.e17-04-0263
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发表时间：
2017-08-09
影响因子：
3.3
通讯作者：
Peterson JR
中科院分区：
文献类型：
--
作者：
Anthony SA;Burrell AL;Johnson MC;Duong-Ly KC;Kuo YM;Simonet JC;Michener P;Andrews A;Kollman JM;Peterson JR
代谢酶IMPDH组装成八聚体，八聚体可以在细胞中聚合并形成微米级结构。八聚体可以采用由变构核苷酸和底物结合驱动的活性的、扩展的或非活性的、压缩的构象。这两种形式都容纳在聚合物中，单独的聚合不会改变催化活性。几种代谢酶进行可逆聚合成大分子组装体。这些组件的功能往往是不清楚的，但在某些情况下，他们调节酶的活性和代谢稳态。鸟嘌呤核苷酸生物合成酶肌苷一磷酸脱氢酶（IMPDH）形成八聚体，其折叠成螺旋链。在哺乳动物细胞中，IMPDH细丝可以结合成微米长度的组装体。聚合和酶活性部分地通过嘌呤核苷酸与变构调节结构域的结合来调节。ATP促进八聚体聚合，而鸟苷三磷酸（GTP）促进一个紧凑的，无活性的构象，其能力是未知的。同样不清楚的是聚合是否直接改变IMPDH催化活性。为了解决这一问题，我们鉴定了人IMPDH 2的阻止或促进聚合的点突变体。出乎意料的是，我们发现聚合和非组装形式的重组IMPDH具有相当的催化活性，底物亲和力和GTP敏感性，并在细胞中验证了这一发现。电子显微镜显示，基板和变构核苷酸移动的八聚体和长丝的活性和非活性构象之间的平衡。与选择性稳定活性或非活性构象的其他代谢细丝不同，重组IMPDH细丝适应多种状态。这些构象状态被底物可用性和嘌呤平衡精细地调节，而聚合可以允许状态之间的合作转换。
The metabolic enzyme IMPDH assembles into octamers that can polymerize and form micron-scale structures in cells. Octamers can adopt active, expanded or inactive, compressed conformations driven by allosteric nucleotide and substrate binding. Both forms are accommodated within polymers, and polymerization alone does not alter catalytic activity. Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear, but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length assemblies. Polymerization and enzyme activity are regulated in part by binding of purine nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, whereas guanosine triphosphate (GTP) promotes a compact, inactive conformation whose ability to polymerize is unknown. Also unclear is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and nonassembled forms of recombinant IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament. Unlike other metabolic filaments, which selectively stabilize active or inactive conformations, recombinant IMPDH filaments accommodate multiple states. These conformational states are finely tuned by substrate availability and purine balance, while polymerization may allow cooperative transitions between states.