基于多位点模型探索葡萄糖醛酸转移代谢非典型动力学的机制

Atypical kinetics represent as common cases in glucuronidation pathway, which makes it sometimes hard to conduct the related researches. Based on multiple site model, this project will explore key chemical structural characteristics which can be recognized by stimulating or inhibitory sites in UDP-glucuronosyltransferases (UGT), by integrating strategies of enzyme kinetics, organic synthesis and computational biology. The results will be further used to explain the mechanisms of atypical kinetics. For the inhibitory site, conclusion will be from studies focused on UGT2B7 mediated glcuronidation exhibiting substrate inhibition kinetics. By performing analogy analysis, chemical structure types prone to substrate inhibition kinetics will be drawn. By conducting regression analysis, quantitative relation between kinetic constants and structure will be modeled. By performing enzyme kinetic analysis, it will be judged whether substrates can bind into C-terminal domain. By utilization of computing software, it will be further revealed the binding pattern between substrates and C-terminal domain. For the stimulating site, related studies will be focused on activation of UGT1A4. A series of diethylstilbestrol analogs will derive from addition reaction and etherification. Kinetic assays will be conducted for the stimulation on UGT1A4. By performing intermolecular superimposition, the key structural information which can result in activation of UGT1A4 will be further extracted. Successful operation of this project will help deeply understand the function of UGTs, and also has a good value in drug development and in prevention and control of UGT related toxicities.

葡萄糖醛酸结合代谢反应广泛表现出非典型动力学，给该代谢路径的相关研究带来了挑战。本项目根据多位点模型，结合酶动力学分析、有机合成、计算生物学的方法，拟对葡萄糖醛酸转移酶(UGT)抑制和激活位点识别的关键结构特征分别进行探索，从而揭示非典型动力学的机制。对于抑制位点，以UGT2B7介导的底物抑制反应为研究对象：通过类比分析，获取易于表现底物抑制动力学的结构类型；通过回归分析，构建底物结构和底物抑制动力学参数的定量关系；通过动力学分析，判断底物抑制型底物能否和C端结构域结合，并借助计算软件解析二者的结合模式。对于激活位点，以UGT1A4的激活为研究对象：通过加成和醚化方法，合成己烯雌酚的系列类似物；根据其对UGT1A4的激活作用，利用分子叠合策略，提取激活位点识别的关键结构特征。本研究不仅对揭示UGT酶功能具有指导意义，而且能够为药物开发及UGT相关毒性的防控提供理论依据。

葡萄糖醛酸转移酶(UGT)是人体最为重要的生化保护屏障，其介导了众多临床药物、环境毒物、内源激素的代谢反应。非典型动力学是该代谢路径中常见的动力学模型，给代谢路径的酶归属、体外体内关联等带来了麻烦。然而，非典型动力学出现的机制一直不为人知，本项目为揭示其背后的机制及影响因素，开展了系列研究工作。针对底物抑制动力学机制，我们首先研究了SN-38的代谢动力学，结果表明牛血清白蛋白可以封闭抑制位点，底物抑制动力学继而演化为米氏动力学。我们随后系统研究了己烯雌酚对UGT酶的抑制作用，结果证实UGT2B7的抑制位点能够结合己烯雌酚，结合能力远超一般底物位点。我们同时研究了厚朴酚对UGT1A9的抑制动力学，结果表明厚朴酚同样能够结合至酶的两个位点，两个位点对厚朴酚的亲和力不同。为深入探讨抑制位点和一般底物结合位点在识别结合配体上的区别，我们开展了系列联苯酚类似物的代谢动力学研究，结果表明反应位点局部强极性对接受葡萄糖醛酸基团不利，相对于底物结合位点，抑制位点倾向于结合疏水性更强的配体。针对激活位点，我们深入研究了双酚A对UGT1A9的影响，结果表明双酚A在低浓度时能提高酶的催化活性，促进作用得益于双酚A能够同时提高酶和糖基供体的结合效率以及催化常数。通过本项目的开展，我们证实UGT酶中存在其它功能位点是非典型动力学产生的原因。而且，抑制位点对配体的疏水性要求较高，并且可以被大分子等封闭；激活位点结合配体后，会对UGT酶的C端结构域的构象产生较大影响。