Wilson R Hunter, Zamfir Serban, Sumner Isaiah
Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, VA 22807, USA.
Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, VA 22807, USA.
J Mol Graph Model. 2017 Sep;76:403-411. doi: 10.1016/j.jmgm.2017.07.006. Epub 2017 Jul 10.
The role of a highly conserved active site asparagine (N79) in the ubiquitin conjugating enzyme, Ubc13, is probed using molecular dynamics simulations. Both wild type and mutant enzymes (N79A and N79D) are studied. Contrary to a popular hypothesis, we show that it is unlikely that N79 stabilizes a reaction intermediate, but instead preferentially hydrogen bonds to a loop near the active site. This keeps the sidechain carboxylate of an aspartate in the loop (D119) near the sidechain amine of the substrate lysine. Our simulations show that this distance increases in the mutants. D119 has been hypothesized to play a variety of roles in the enzyme, including deprotonating the substrate lysine, so changing this distance can have an effect on the enzyme's efficiency. Finally, we show that it is possible for the aspartate to deprotonate the substrate even across long distances if short water wires form that connect the proton donor and acceptor. Short water wires form with greater probability in the wild type than in mutant enzymes.
利用分子动力学模拟探究了泛素结合酶Ubc13中一个高度保守的活性位点天冬酰胺（N79）的作用。研究了野生型和突变型酶（N79A和N79D）。与一个流行的假设相反，我们发现N79不太可能稳定反应中间体，而是优先与活性位点附近的一个环形成氢键。这使得环中天冬氨酸（D119）的侧链羧酸盐靠近底物赖氨酸的侧链胺。我们的模拟表明，在突变体中这个距离会增加。有人假设D119在该酶中发挥多种作用，包括使底物赖氨酸去质子化，因此改变这个距离可能会影响酶的效率。最后，我们表明，如果形成连接质子供体和受体的短水线，天冬氨酸甚至可以在远距离使底物去质子化。短水线在野生型中形成的概率比在突变型酶中更高。
