The Lysine Deprotonation Mechanism in a Ubiquitin Conjugating Enzyme

Ubiquitination is a biochemical reaction in which a small
protein,
ubiquitin (Ub), is covalently linked to a lysine on a target protein.
This type of post-translational modification can signal for protein
degradation, DNA repair, or inflammation response. Ubiquitination
is catalyzed by three families of enzymes: ubiquitin activating enzymes
(E1), ubiquitin conjugating enzymes (E2), and ubiquitin ligases (E3).
In this study, we focus on the chemical mechanism used by the E2 enzyme,
Ubc13, which forms polyubiquitin chains by linking a substrate Ub
to Lys63 on a target ubiquitin (Ub*). Initially, Ubc13 is covalently
linked to the substrate Ub. Next, Lys63 in the Ub* is deprotonated,
becomes an active nucleophile, and attacks the thioester bond in the
Ubc13∼Ub conjugate. The deprotonation mechanism is not well
understood. There are two, conserved nearby residues that may act
as conjugate bases (Asp119 on Ubc13 and Glu64 on Ub*.) It is also
hypothesized that the active site environment suppresses the lysine’s
pKa, favoring deprotonated lysine. We
test these hypotheses by simulating both WT and mutant Ubc13 with
constant pH molecular dynamics (CpHMD), which allows titratable residues
to change their protonation states. In our simulations, we have five
titratable residues, including Lys63, and we use these simulations
to monitor the protonation states and to generate titration curves
of lysine 63. We found that the pKa of
Lys63 is highly dependent on its distance from the active site. Also,
mutating Asp119 or Glu64 to Ala has little effect on the lysine pKa, indicating that neither residue acts as a
generalized base. Finally, we note that mutating a structural residue
(Asn79 to Ala) increases the lysine pKa, suggesting that alterations to the active site hydrogen bonding
network can affect nucleophile activation.

泛素化（Ubiquitination）是一类生化反应，指小分子蛋白质泛素（ubiquitin, Ub）与靶蛋白的赖氨酸发生共价结合的过程。此类翻译后修饰（post-translational modification）可介导蛋白质降解、DNA修复或炎症应答。泛素化由三类酶催化：泛素激活酶（ubiquitin activating enzymes, E1）、泛素结合酶（ubiquitin conjugating enzymes, E2）与泛素连接酶（ubiquitin ligases, E3）。本研究聚焦于E2酶Ubc13所采用的化学机制：该酶通过将底物泛素连接至靶泛素（Ub*）的Lys63位点，进而形成多泛素链（polyubiquitin chains）。反应初始阶段，Ubc13与底物泛素形成共价结合物。随后，Ub*中的Lys63发生去质子化（deprotonation），转变为活性亲核试剂（nucleophile），并攻击Ubc13~Ub结合物中的硫酯键（thioester bond）。目前该去质子化机制尚未被充分阐明。已知存在两个保守的邻近残基可能作为共轭碱（conjugate bases）：Ubc13上的Asp119与Ub*上的Glu64。另有假说提出，活性位点微环境可降低赖氨酸的pKa，更有利于其去质子化状态的形成。本研究通过恒pH分子动力学（constant pH molecular dynamics, CpHMD）模拟野生型与突变型Ubc13，以验证上述假说——该方法可使可滴定残基（titratable residues）改变其质子化状态（protonation states）。本次模拟共包含包括Lys63在内的5个可滴定残基，我们通过这些模拟监测质子化状态，并生成Lys63的滴定曲线（titration curves）。研究结果显示，Lys63的pKa高度依赖其与活性位点的距离。此外，将Asp119或Glu64突变为丙氨酸（Ala）对赖氨酸的pKa几乎无影响，表明这两个残基均未起到通用碱（generalized base）的作用。最后，我们注意到将结构残基Asn79突变为丙氨酸（Ala）会升高赖氨酸的pKa，这提示活性位点氢键网络（hydrogen bonding network）的改变可影响亲核试剂的活化。