近日，中國科學(xué)院上海藥物研究所蔣華良課題組在《科學(xué)公共圖書館—綜合(PLoS ONE)》上發(fā)表了關(guān)于葡萄糖激酶催化機(jī)制研究的論文（PLoS ONE, 2009, 7: e6304）,，闡述了葡萄糖激酶（Glucokinase, GK）催化過程的分子機(jī)理,。
葡萄糖激酶是調(diào)節(jié)人體血液中葡萄糖水平的重要酶，主要作用是監(jiān)控血中的葡萄糖水平,。臨床上，由于葡萄糖激酶過度激活或者失活，導(dǎo)致血液中葡萄糖水平過低或過高,，繼而引發(fā)2型糖尿病和高血糖癥病變。因此，以葡萄糖激酶為靶標(biāo)的抗糖尿病研發(fā)引起了各界的廣泛關(guān)注,。對(duì)于葡萄糖激酶臨床突變研究和以葡萄糖激酶為靶標(biāo)的藥物開發(fā)來說,，靶標(biāo)自身功能機(jī)制的闡述是亟需解決的重要問題之一。由于葡萄糖激酶催化葡萄糖磷酸化時(shí)需要大規(guī)模的構(gòu)象變化,，現(xiàn)有的實(shí)驗(yàn)方法還不能有效地檢測這些變化,，同時(shí)葡萄糖激酶-ATP-葡萄糖三元復(fù)合物晶體結(jié)構(gòu)很難測定。在葡萄糖激酶—構(gòu)機(jī)制研究的基礎(chǔ)上(PNAS 2006, 103, 13368-13373),，蔣華良研究員帶領(lǐng)研究生張健等綜合利用計(jì)算生物學(xué)和分子生物學(xué)方法,，對(duì)葡萄糖催化的機(jī)制進(jìn)行了深入系統(tǒng)的研究。采用分子模擬和分子動(dòng)力學(xué)方法在構(gòu)建了葡萄糖激酶-ATP-葡萄糖三元復(fù)合物的三維結(jié)構(gòu),，獲得了精確的葡萄糖激酶的催化反應(yīng)環(huán)境,，發(fā)現(xiàn)了與葡萄糖激酶催化活性密切相關(guān)的一系列重要?dú)埢渲蠰ys169殘基在底物ATP和葡萄糖的結(jié)合中發(fā)揮決定性作用,，首次合理地解釋了臨床上常見的K169N缺陷型葡萄糖激酶突變體的分子機(jī)理,。與沈旭研究員課題組合作，用分子生物學(xué)實(shí)驗(yàn)和酶動(dòng)力學(xué)學(xué)分析方法驗(yàn)證了理論計(jì)算結(jié)果(這部分工作主要由黎陳靜完成),。繼而,，博士后石婷應(yīng)用量子力學(xué)/分子力學(xué)(QM/MM)相結(jié)合的方法，在原子水平上研究了葡萄糖激酶的催化機(jī)制,，發(fā)現(xiàn)Lys169在扮演酸催化劑的作用,。該研究為進(jìn)一步開展臨床上缺陷型葡萄糖激酶突變體的治療以及設(shè)計(jì)抗糖尿病藥物提供了重要信息，也是上海藥物所藥物發(fā)現(xiàn)與設(shè)計(jì)中心用理論計(jì)算與實(shí)驗(yàn)相結(jié)合的方法研究生物學(xué)問題的又一成功案例,。
該研究項(xiàng)目得到了國家科技部,、基金委、上海市科委和中科院的資助,。（生物谷Bioon.com）
生物谷推薦原始出處：
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.
