Phosphoryl Transfers of the Phospholipase D Superfamily: A Quantum Mechanical Theoretical Study
Phosphoryl Transfers of the Phospholipase D Superfamily: A Quantum Mechanical Theoretical Study
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DOI：
10.1021/ja4042753
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发表时间：
2013-09-18
影响因子：
15
通讯作者：
Webster, Charles Edwin
中科院分区：
文献类型：
--
作者：
DeYonker, Nathan J.;Webster, Charles Edwin
含HKD的磷脂酶D超家族催化磷脂酰胆碱的头基裂解以产生磷脂酸和胆碱。从理论上研究了这种解理过程的机理。我们的模型的几何基础是从链霉菌菌株PMF（PDB代码= 1V 0 Y）的五坐标磷酸组氨酸中间体的X-射线晶体结构。混合ONIOM QM：QM方法与密度泛函理论（DFT）和半经验PM 6（DFT：PM 6）是用来获取热力学和动力学数据的初始磷酰基转移，随后的水解，最后，形成实验观察到的“死端”磷酸组氨酸产品（PDB代码= 1V 0 W）。该模型包含十九个氨基酸残基（包括两个高度保守的HKD基序），四个明确的水分子，和底物。通过计算，短寿命的五配位正膦中间体在分钟时间尺度上的持久性是合理的。这种五配位磷酸组氨酸中间体在水解事件和“底物重组”（活性位点内体外模型底物的重组）之间存在。计算结果直接支持了体外四配位磷酸组氨酸产物的热力学稳定性。在体内，底物重组的活化能太高，可能是由于包埋在脂质双层中时底物不动性的组合，以及与体外底物浸泡中使用的化合物相比其更大的空间体积。在这个较长的时间范围内，酶将沿着脂膜向其下一个底物靶点迁移，而不是促进死端产物的形成。
The HKD-containing Phospholipase D superfamily catalyzes the cleavage of the headgroup of phosphatidylcholine to produce phosphatidic acid and choline. The mechanism of this cleavage process is studied theoretically. The geometric basis of our models is the X-ray crystal structure of the five-coordinate phosphohistidine intermediate from Streptomyces sp. Strain PMF (PDB Code = 1V0Y). Hybrid ONIOM QM:QM methodology with Density Functional Theory (DFT) and semiempirical PM6 (DFT:PM6) is used to acquire thermodynamic and kinetic data for the initial phosphoryl transfer, subsequent hydrolysis, and finally, the formation of the experimentally observed "dead-end" phosphohistidine product (PDB Code = 1V0W). The model contains nineteen amino acid residues (including the two highly conserved HKD-motifs), four explicit water molecules, and the substrate. Via computations, the persistence of the short-lived five-coordinate phosphorane intermediate on the minutes times scale is rationalized. This five-coordinate phosphohistidine intermediate energetically exists between the hydrolysis event and "substrate reorganization" (the reorganization of the in vitro model substrate within the active site). Computations directly support the thermodynamic favorability of the in vitro four-coordinate phosphohistidine product. In vivo, the activation energy of substrate reorganization is too high, perhaps due to a combination of substrate immobility when embedded in the lipid bilayer, as well as its larger steric bulk compared to the compound used in the in vitro substrate soaks. On this longer time scale, the enzyme will migrate along the lipid membrane toward its next substrate target, rather than promote the formation of the dead-end product.