Preorganized Internal Electric Field Promotes a Double-Displacement Mechanism for the Adenine Excision Reaction by Adenine DNA Glycosylase

Adenine DNA glycosylase (MutY) is a monofunctional glycosylase,
removing adenines (A) misinserted opposite 8-oxo-7,8-dihydroguanine
(OG), a common product of oxidative damage to DNA. Through multiscale
calculations, we decipher a detailed adenine excision mechanism of
MutY that is consistent with all available experimental data, involving
an initial protonation step and two nucleophilic displacement steps.
During the first displacement step, N-glycosidic bond cleavage is
accompanied by the attack of the carboxylate group of residue Asp144
at the anomeric carbon (C1′), forming a covalent glycosyl-enzyme
intermediate to stabilize the fleeting oxocarbenium ion. After departure
of the excised base, water nucleophiles can be recruited to displace
Asp144, completing the catalytic cycle with retention of stereochemistry
at the C1′ position. The two displacement reactions are found
to mostly involve the movement of the oxocarbenium ion, occurring
with large charge reorganization and thus sensitive to the internal
electric field (IEF) exerted by the polar protein environment. Intriguingly,
we find that the negatively charged carboxylate group is a good nucleophile
for the oxocarbenium ion, yet an unactivated water molecule is not,
and that the electric field catalysis strategy is used by the enzyme
to enable its unique double-displacement reaction mechanism. A strong
IEF, pointing toward 5′ direction of the substrate sugar ring,
greatly facilitates the second displacement reaction at the expense
of elevating the barrier of the first one, thereby allowing both reactions
to occur. These findings not only increase our understanding of the
strategies used by DNA glycosylases to repair DNA lesions, but also
have important implications for how internal/external electric field
can be applied to modulate chemical reactions.

腺嘌呤DNA糖苷酶（Adenine DNA glycosylase，MutY）是一类单功能糖苷酶，可移除插入至8-氧代-7,8-二氢鸟嘌呤（8-oxo-7,8-dihydroguanine，OG，DNA氧化损伤的常见产物）对面的腺嘌呤（A）。本研究通过多尺度计算，解析了MutY完整且与所有现有实验数据相符的腺嘌呤切除机制，该机制包含初始质子化步骤与两次亲核取代步骤。在首次取代步骤中，N-糖苷键的断裂伴随天冬氨酸144（Asp144）残基的羧基基团进攻异头碳（C1′），形成共价糖苷-酶中间体以稳定短暂存在的氧鎓离子。切除的碱基脱离后，可招募水亲核试剂取代Asp144，在保留C1′位立体化学构型的前提下完成催化循环。研究发现，两次取代反应主要涉及氧鎓离子的移动，该过程伴随显著的电荷重组，因此对极性蛋白质环境产生的内部电场（internal electric field，IEF）极为敏感。有趣的是，本研究发现带负电的羧基基团可作为氧鎓离子的优质亲核试剂，而未活化的水分子则不具备该能力；同时该酶借助电场催化策略实现其独特的双取代反应机制。指向底物糖环5′方向的强内部电场，虽会升高首次取代反应的能垒，却可极大促进第二次取代反应，最终使两次反应均能顺利进行。本研究结果不仅加深了我们对DNA糖苷酶修复DNA损伤策略的认知，也为利用内/外部电场调控化学反应提供了重要的理论参考。