Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička 54, Zagreb, 10000, Croatia.
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, Warsaw, 02-093, Poland.
Sci Rep. 2018 Oct 18;8(1):15427. doi: 10.1038/s41598-018-33723-1.
Purine nucleoside phosphorylase (PNP) catalyses the cleavage of the glycosidic bond of purine nucleosides using phosphate instead of water as a second substrate. PNP from Escherichia coli is a homohexamer, build as a trimer of dimers, and each subunit can be in two conformations, open or closed. This conformational change is induced by the presence of phosphate substrate, and very likely a required step for the catalysis. Closing one active site strongly affects the others, by a yet unclear mechanism and order of events. Kinetic and ligand binding studies show strong negative cooperativity between subunits. Here, for the first time, we managed to monitor the sequence of nucleoside binding to individual subunits in the crystal structures of the wild-type enzyme, showing that first the closed sites, not the open ones, are occupied by the nucleoside. However, two mutations within the active site, Asp204Ala/Arg217Ala, are enough not only to significantly reduce the effectiveness of the enzyme, but also reverse the sequence of the nucleoside binding. In the mutant the open sites, neighbours in a dimer of those in the closed conformation, are occupied as first. This demonstrates how important for the effective catalysis of Escherichia coli PNP is proper subunit cooperation.
嘌呤核苷磷酸化酶（PNP）使用磷酸盐而不是水作为第二底物催化嘌呤核苷的糖苷键断裂。大肠杆菌中的 PNP 是一种同六聚体，由三聚体二聚体组成，每个亚基可以处于两种构象，开放或关闭。这种构象变化是由磷酸盐底物的存在诱导的，很可能是催化所必需的步骤。通过一个尚未阐明的机制和事件顺序，关闭一个活性位点强烈影响其他活性位点。动力学和配体结合研究表明亚基之间存在强烈的负协同作用。在这里，我们首次成功地在野生型酶的晶体结构中监测到核苷结合到各个亚基的顺序，结果表明首先是封闭的位点，而不是开放的位点，被核苷占据。然而，在活性位点内的两个突变，天冬氨酸 204 到丙氨酸/精氨酸 217 到丙氨酸，不仅足以显著降低酶的效率，而且还可以逆转核苷结合的顺序。在突变体中，开放的位点，与处于封闭构象的二聚体中的那些相邻的位点，首先被占据。这证明了大肠杆菌 PNP 的有效催化对于适当的亚基合作是多么重要。
