烟酰胺型辅酶性质、修饰及分子对接研究
文献类型：学位论文
| 作者 | 马洪静 |
| 学位类别 | 硕士 |
| 答辩日期 | 2005-05-26 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 过程工程研究所 |
| 导师 | 王平 |
| 关键词 | 8-(6-氨基己烷)-氨基-NADH 辅酶修饰 辅酶稳定性 分子对接 |
| 学位专业 | 生物化工 |
| 中文摘要 | 辅酶依赖型氧化还原酶类所催化的反应在制备手性醇、羟基酸、氨基酸方面具有广泛的应用。在实际应用中，辅酶通常需要连接在大分子化合物上或者固相载体上以便重复使用和循环再生，而采用适当的分子对辅酶进行化学修饰以引入较活泼的反应基团和间隔臂通常是重要的步骤。 本文第一部分首先对天然辅酶的稳定性和与载体连接的反应性做了初步研究。NADH在中酸性环境中(pH 2.2-8.0)，失活符合一级反应公式，速率随pH降低而增大。NAD+在碱性条件下易发生降解反应。分别尝试用带有环氧基，氨基和羧基的纳米颗粒对辅酶进行固定化，结果表明辅酶的反应活性较差，均未获得固定量和生物活性。 本文第二部分采用己二胺对辅酶进行定点修饰，通过DEAE-Sepharose层析分离得到产物8-(6-氨基己烷)-氨基-NADH (NH2(CH2)6NH-NADH)。在pH 2.2-8.0范围内系统考察了修饰产物的pH稳定性，结果表明在不同种类的缓冲液中，修饰后辅酶的稳定性均有显著提高。生物活性研究表明，对乳酸脱氢酶(LDH)体系，在底物丙酮酸过量(10mM)的条件下，以NH2(CH2)6NH-NADH为辅酶测得的反应速率为同样浓度NADH的3.0-3.8倍。对谷氨酸脱氢酶(GDH)体系，当底物NH4+和-酮戊二酸的浓度分别为50mM和10mM时(过量)，NH2(CH2)6NH-NADH的速率是NADH 1.3-1.4倍。 本文第三部分用乙醇脱氢酶(ADH)和乳酸脱氢酶(LDH)对修饰前后辅酶的全动力学参数进行了测定，并用AutoDock分子对接软件模拟了辅酶和酶分子结合。分子对接发现NH2(CH2)6NH-NADH可以和ADH的Glu33残基形成一个额外的氢键，并且与ADH和LDH的氨基酸残基形成额外的疏水作用，而且与酶分子的结合自由能△G要低于NADH。这一结果与全动力学实验所测得的修饰后辅酶与LDH和ADH具有较小的解离常数Kia值这一结果相符，说明NH2(CH2)6NH-NADH和两种酶的结合都更加容易。但是修饰后辅酶对酶与底物的结合却产生了不同的影响：对于ADH，底物乙醛的米式常数Km值减小；对LDH，底物丙酮酸的Km值增加。 本研究成功制备了一种高pH稳定性和活性的辅酶衍生物NH2(CH2)6NH-NADH，并首次采用分子对接方法对NH2(CH2)6NH-NADH和乳酸脱氢酶/乙醇脱氢酶的结合进行了模拟，在分子水平揭示了修饰后辅酶的结构对活性的影响。 |
| 英文摘要 | Coenzyme dependent dehydrogenases are widely used for the biosyntheses of chiral alcohols, hydroxy acids, amino acid. For most practical uses, cofactors need to be linked to polymers or attached to solid carriers to facilitate the reuse and regeneration. Before macromolecularization or immobilization, chemical modification of coenzyme is usually the primary step to introduce spacer arm and reactive functional group. In the first part, we systematically investigated the pH stability of native coenzyme and its reactivity with solid carrier. Under acid or neutral conditions, the deactivation rate of NADH obeys a first-order reaction formula and increased with decrease of pH. For NAD+, the deactivation rate increased with the increase of pH under alkaline condition. Nanoparticles with epoxy group, amino group or carboxy group were used for the immobilization of coenzyme. Due to the poor reactivity of coenzyme, no deteactable immobilization amount and activity were observed. In the second part, we prepared 8-(6-aminohexyl)-amino-NADH by modifying NAD+ with hexanediamine at the C-8 position of adenine moiety. After 3-steps synthesizing process, the product was purified on DEAE-Sepharose column. The pH stability at acid or neutral environments was studied and compared with NADH. The results showed that the modified NADH was more stable than the native one. Catalytic activity of the chemical modified coenzyme was evaluated with lactate dehydrogenase (LDH) and glutamate dehydrogenase (GDH) systems. For LDH (pyruvic acid, 10mM) and GDH (NH4+, 50mM;-ketoglutaric acid，10mM), the activity for NH2(CH2)6NH-NADH is 3.0-3.8 or 1.3-1.4 times higher than that of native NADH, respectively. In the last part, in order to investigate the effect of modification on the activity of coenzyme, we determined the full-kinetic parameters for NH2(CH2)6NH-NADH and NADH with alcohol dehydrogenase (ADH) and LDH, and carried out molecule docking experiment by using the Autodock 4.0 to get a insight to the coenzyme binding at the active site of enzyme. The docking results showed that hexanediamine arm can form an extra hydrogen bond with Glu33 in ADH and more hydrophobic interactions with surrounding amino acids in both ADH and LDH, which consequently made the binding energy lower. This result was in accordance with the change tendency of the dissociation constants (Kia) for coenzyme-enzyme complexes obtained from full-kinetic experiments for both ADH and LDH. The hexanediamine arm affected the binding of substrate to enzyme-coenzyme complex differently: for ADH, the Michaelis constant (Km) for aldehyde decreased, while for LDH, the Km value for pyruvate acid increased. In this thesis, we successfully synthesized a coenzyme derivative carryinng a functional group suitable for further derivation with enhanced pH stability and activity, and it is for the fist time to interpret the interactions of NH2(CH2)6NH-NADH with the active site of ADH and LDH using molecule docking methods. |
| 语种 | 中文 |
| 公开日期 | 2013-09-16 |
| 页码 | 91 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1292]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 马洪静. 烟酰胺型辅酶性质、修饰及分子对接研究[D]. 过程工程研究所. 中国科学院过程工程研究所. 2005. |
入库方式： OAI收割
来源：过程工程研究所
浏览0
下载0
收藏0
其他版本
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。