Tomić Antonija, Tomić Sanja
Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička 54, 10 000 Zagreb, Croatia.
Dalton Trans. 2014 Nov 7;43(41):15503-14. doi: 10.1039/c4dt02003k.
Multiple choices of the protein active conformations in flexible metalloenzymes complicate study of their catalytic mechanism. We used three different conformations of human dipeptidyl-peptidase III (DPP III) to investigate the influence of the protein environment on ligand binding and the Zn(2+) coordination. MD simulations followed by calculations of binding free energy components accomplished for a series of DPP III substrates, both synthetic and natural, revealed that binding of the β-strand shaped substrate to the five-stranded β-core of the compact DPP III form (in antiparallel fashion) is the preferred binding mode, in agreement with the experimentally determined structure of the DPP III inactive mutant-tynorphin complex (Bezerra et al., Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 6525). Previously it was proposed that the catalytic mechanism of DPP III is similar to that of thermolysin, which assumes exchange of five and four coordinated Zn(2+), and activation of Zn-bound water by a nearby Glu. Our QM/MM calculations, performed for a total of 18 protein structures with different zinc ion environments, revealed that the 5-coordinated metal ion is more favourable than the 6-coordinated one in only the most compact DPP III form. Besides, in this structure E451 is H-bonded to the metal ion coordinating water. Also, our study revealed two constraints for the broad substrate specificity of DPP III. One is the possibility of the substrate adopting the β-strand shape and the other is its charged N-terminus. Altogether, we assume that the human DPP III active conformation would be the most compact form, similar to the "closed X-ray" DPP III structure.
柔性金属酶中蛋白质活性构象的多种选择使其催化机制的研究变得复杂。我们使用人二肽基肽酶III（DPP III）的三种不同构象来研究蛋白质环境对配体结合和Zn(2+)配位的影响。对一系列合成和天然的DPP III底物进行分子动力学（MD）模拟，随后计算结合自由能成分，结果表明β链状底物以反平行方式与紧密型DPP III形式的五链β核心结合是首选的结合模式，这与DPP III无活性突变体-酪氨啡肽复合物的实验测定结构一致（Bezerra等人，《美国国家科学院院刊》，2012年，109卷，6525页）。此前有人提出DPP III的催化机制与嗜热菌蛋白酶相似，即假定五配位和四配位的Zn(2+)发生交换，且附近的谷氨酸激活与锌结合的水。我们对总共18种具有不同锌离子环境的蛋白质结构进行的量子力学/分子力学（QM/MM）计算表明，仅在最紧密的DPP III形式中，五配位金属离子比六配位金属离子更有利。此外，在这种结构中，E451与配位水形成氢键。而且，我们的研究揭示了DPP III具有广泛底物特异性的两个限制因素。一个是底物形成β链形状的可能性，另一个是其带电荷的N末端。总之，我们推测人DPP III的活性构象将是最紧密的形式，类似于“封闭的X射线”DPP III结构。
