Pyruvate Formate-lyase, Evidence for an Open Conformation Favored in the Presence of Its Activating Enzyme
Pyruvate Formate-lyase, Evidence for an Open Conformation Favored in the Presence of Its Activating Enzyme
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DOI：
10.1074/jbc.m109.096875
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发表时间：
2010-08-27
影响因子：
4.8
通讯作者：
Broderick, Joan B.
中科院分区：
文献类型：
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作者：
Peng, Yi;Veneziano, Susan E.;Broderick, Joan B.
丙酮酸甲酸裂解酶激活酶（PFL-AE）通过在丙酮酸甲酸裂解酶（PFL）的Gly-734位上产生催化必需的自由基来激活PFL。未活化的PFL的晶体结构显示，Gly-734被埋在距离蛋白质表面8埃的地方，我们在这里称之为PFL的闭合构象。我们在这里提供了第一个实验证据的PFL的一个替代的开放构象，其中：（i）甘氨酰自由基是显着不太稳定;（ii）活化的酶表现出较低的催化活性;（iii）甘氨酰自由基进行较少的H/D交换与溶剂;和（iv）T-m的蛋白质降低。证据表明，在PFL的开放构象中，Gly-734残基不是位于其在酶活性位点中的掩埋位置，而是位于更多溶剂暴露的位置。此外，我们发现PFL-AE的存在增加了PFL在开放构象中的比例;这一观察结果支持PFL-AE通过与开放构象中的Gly-734环结合来访问Gly-734以直接夺取氢原子的想法，从而将PFL的闭合7开放平衡向右移动。总之，我们的研究结果导致一个模型，其中PFL可以存在于一个封闭的构象，与Gly-734埋在PFL的活性位点和窝藏一个稳定的甘氨酰基自由基，或开放的构象，与Gly-734更多的溶剂暴露和访问的PFL-AE活性位点。这两种构象之间的平衡PFL调制与PFL-AE的相互作用。
Pyruvate formate-lyase-activating enzyme (PFL-AE) activates pyruvate formate-lyase (PFL) by generating a catalytically essential radical on Gly-734 of PFL. Crystal structures of unactivated PFL reveal that Gly-734 is buried 8 angstrom from the surface of the protein in what we refer to here as the closed conformation of PFL. We provide here the first experimental evidence for an alternate open conformation of PFL in which: (i) the glycyl radical is significantly less stable; (ii) the activated enzyme exhibits lower catalytic activity; (iii) the glycyl radical undergoes less H/D exchange with solvent; and (iv) the T-m of the protein is decreased. The evidence suggests that in the open conformation of PFL, the Gly-734 residue is located not in its buried position in the enzyme active site but rather in a more solvent-exposed location. Further, we find that the presence of the PFL-AE increases the proportion of PFL in the open conformation; this observation supports the idea that PFL-AE accesses Gly-734 for direct hydrogen atom abstraction by binding to the Gly-734 loop in the open conformation, thereby shifting the closed 7 open equilibrium of PFL to the right. Together, our results lead to a model in which PFL can exist in either a closed conformation, with Gly-734 buried in the active site of PFL and harboring a stable glycyl radical, or an open conformation, with Gly-734 more solvent-exposed and accessible to the PFL-AE active site. The equilibrium between these two conformations of PFL is modulated by the interaction with PFL-AE.