近日，中国科学院上海药物研究所蒋华良课题组在《科学公共图书馆—综合(PLoS ONE)》上发表了关于葡萄糖激酶催化机制研究的论文（PLoS ONE, 2009, 7: e6304），阐述了葡萄糖激酶（Glucokinase, GK）催化过程的分子机理。
葡萄糖激酶是调节人体血液中葡萄糖水平的重要酶，主要作用是监控血中的葡萄糖水平。临床上，由于葡萄糖激酶过度激活或者失活，导致血液中葡萄糖水平过低或过高，继而引发2型糖尿病和高血糖症病变。因此，以葡萄糖激酶为靶标的抗糖尿病研发引起了各界的广泛关注。对于葡萄糖激酶临床突变研究和以葡萄糖激酶为靶标的药物开发来说，靶标自身功能机制的阐述是亟需解决的重要问题之一。由于葡萄糖激酶催化葡萄糖磷酸化时需要大规模的构象变化，现有的实验方法还不能有效地检测这些变化，同时葡萄糖激酶-ATP-葡萄糖三元复合物晶体结构很难测定。在葡萄糖激酶—构机制研究的基础上(PNAS 2006, 103, 13368-13373)，蒋华良研究员带领研究生张健等综合利用计算生物学和分子生物学方法，对葡萄糖催化的机制进行了深入系统的研究。采用分子模拟和分子动力学方法在构建了葡萄糖激酶-ATP-葡萄糖三元复合物的三维结构，获得了精确的葡萄糖激酶的催化反应环境，发现了与葡萄糖激酶催化活性密切相关的一系列重要残基，其中Lys169残基在底物ATP和葡萄糖的结合中发挥决定性作用，首次合理地解释了临床上常见的K169N缺陷型葡萄糖激酶突变体的分子机理。与沈旭研究员课题组合作，用分子生物学实验和酶动力学学分析方法验证了理论计算结果(这部分工作主要由黎陈静完成)。继而，博士后石婷应用量子力学/分子力学(QM/MM)相结合的方法，在原子水平上研究了葡萄糖激酶的催化机制，发现Lys169在扮演酸催化剂的作用。该研究为进一步开展临床上缺陷型葡萄糖激酶突变体的治疗以及设计抗糖尿病药物提供了重要信息，也是上海药物所药物发现与设计中心用理论计算与实验相结合的方法研究生物学问题的又一成功案例。
该研究项目得到了国家科技部、基金委、上海市科委和中科院的资助。（生物谷Bioon.com）
生物谷推荐原始出处：
PLoS ONE 4(7): e6304. doi:10.1371/journal.pone.0006304
Lys169 of Human Glucokinase Is a Determinant for Glucose Phosphorylation: Implication for the Atomic Mechanism of Glucokinase Catalysis
Jian Zhang1#, Chenjing Li1#, Ting Shi1#, Kaixian Chen1, Xu Shen1,2, Hualiang Jiang1,2*
1 Center for Drug Discovery and Design, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Graduate School of Chinese Academy of Sciences, Shanghai, China, 2 School of Pharmacy, East China University of Science and Technology, Shanghai, China
Glucokinase (GK), a glucose sensor, maintains plasma glucose homeostasis via phosphorylation of glucose and is a potential therapeutic target for treating maturity-onset diabetes of the young (MODY) and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). To characterize the catalytic mechanism of glucose phosphorylation by GK, we combined molecular modeling, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) calculations, experimental mutagenesis and enzymatic kinetic analysis on both wild-type and mutated GK. Our three-dimensional (3D) model of the GK-Mg2+-ATP-glucose (GMAG) complex, is in agreement with a large number of mutagenesis data, and elucidates atomic information of the catalytic site in GK for glucose phosphorylation. A 10-ns MD simulation of the GMAG complex revealed that Lys169 plays a dominant role in glucose phosphorylation. This prediction was verified by experimental mutagenesis of GK (K169A) and enzymatic kinetic analyses of glucose phosphorylation. QM/MM calculations were further used to study the role of Lys169 in the catalytic mechanism of the glucose phosphorylation and we found that Lys169 enhances the binding of GK with both ATP and glucose by serving as a bridge between ATP and glucose. More importantly, Lys169 directly participates in the glucose phosphorylation as a general acid catalyst. Our findings provide mechanistic details of glucose phorphorylation catalyzed by GK, and are important for understanding the pathogenic mechanism of MODY.
