Enhancing allosteric inhibition in Thermus thermophilus Phosphofructokinase.
Enhancing allosteric inhibition in Thermus thermophilus Phosphofructokinase.
复制标题
增强嗜热磷酸果糖激酶中的变构抑制作用。
DOI：
10.1021/bi501127a
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发表时间：
2015-01-27
期刊：
影响因子：
2.9
通讯作者：
Reinhart, Gregory D.
中科院分区：
文献类型：
--
作者：
McGresham, Maria S.;Reinhart, Gregory D.
极端嗜热栖热菌的磷酸果糖激酶（PFK）中底物Fru-6-P和抑制剂磷酸（烯醇）丙酮酸（PEP）的结合之间的偶联比嗜热脂肪芽孢杆菌的PFK中所见的弱得多。从嗜热脂肪芽孢杆菌PFK（BsPFK）的晶体结构来看，BsPFK中59、158和215位的残基位于从变构位点到最近的活性位点的路径上，并且是连接这两个位点的复杂氢键网络的一部分。用BsPFK中这些位置上的氨基酸取代嗜热栖热菌PFK（TtPFK）中的相应残基，使我们能够将PEP的变构抑制提高近3 kcal mol-1（50倍）至大于或等于BsPFK中观察到的偶联的值。有趣的是，每一个单一的变异N59 D，A158 T，和S215 H产生了大约1千卡mol-1的抑制耦合自由能的增加。这些变体的作用在双变体N59 D/A158 T、N59 D/S215 H和A158 T/S215 H的三种组合中以及在三变体N59 D/A158 T/S215 H中基本上是加和的。因此，虽然所确定的氢键网络可能参与抑制性变构通信，但这些数据不支持需要连接这些位点的相互作用链的模型。尽管细菌PFK的变构激活剂MgADP在相同的变构位点结合，但位置59、158和215处的取代对结合亲和力和MgADP的变构激活没有同样显著的影响。S215 H和N59 D/A158 T/S215 H取代对MgADP激活的影响无法确定，因为S215 H取代导致MgADP结合亲和力急剧下降。单一变体N59 D和A158 T支持结合，但与野生型TtPFK相比，MgADP活化的自由能几乎没有变化。这些结果支持以前的建议，异嗜性抑制和激活发生不同的途径原核PFK。
The coupling between the binding of the substrate Fru-6-P and the inhibitor phospho(enol)pyruvate (PEP) in phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus is much weaker than that seen in a PFK from Bacillus stearothermophilus. From the crystal structures of Bacillus stearothermophilus PFK (BsPFK) the residues at positions 59, 158, and 215 in BsPFK are located on the path leading from the allosteric site to the nearest active site and are part of the intricate hydrogen-bonding network connecting the two sites. Substituting the corresponding residues in Thermus thermophilus PFK (TtPFK) with the amino acids found at these positions in BsPFK allowed us to enhance the allosteric inhibition by PEP by nearly 3 kcal mol–1 (50-fold) to a value greater than or equal to the coupling observed in BsPFK. Interestingly, each single variant N59D, A158T, and S215H produced a roughly 1 kcal mol–1 increase in coupling free energy of inhibition. The effects of these variants were essentially additive in the three combinations of double variants N59D/A158T, N59D/S215H, and A158T/S215H as well as in the triple variant N59D/A158T/S215H. Consequently, while the hydrogen-bonding network identified is likely involved in the inhibitory allosteric communication, a model requiring a linked chain of interactions connecting the sites is not supported by these data. Despite the fact that the allosteric activator of the bacterial PFK, MgADP, binds at the same allosteric site, the substitutions at positions 59, 158, and 215 do not have an equally dramatic effect on the binding affinity and the allosteric activation by MgADP. The effect of the S215H and N59D/A158T/S215H substitutions on the activation by MgADP could not be determined because of a dramatic drop in MgADP binding affinity that resulted from the S215H substitution. The single variants N59D and A158T supported binding but showed little change in the free energy of activation by MgADP compared to the wild type TtPFK. These results support previous suggestions that heterotropic inhibition and activation occur by different pathways prokaryotic PFK.
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