Channeling Within Glycolysis: Steps Involving Nicotine Adenine Dinucleotide

描述（由申请人提供）：通道假说（即，底物或辅酶可以从一个活性位点移动到另一个活性位点而不与本体介质平衡）在糖酵解中仍然是有争议的，因为糖酵解酶很大并且具有很难检测的非常动态的相互作用。在三羧酸循环中已经证明了双链化，其中酶-酶复合物已经结晶并且静电沟道途径被清楚地建模。各种实验方法已经提供了证据表明，通道可能发生在糖酵解，包括沉降，共沉淀，和电泳。也有证据表明糖酵解途径中存在代谢子，一种功能性多酶复合物。计算机建模模拟可以展示潜在的酶-酶复合物，并在配体从一个活性位点移动到另一个活性位点时跟踪配体的轨迹。在此，探索糖酵解中的通道假说将开始通过使用布朗动力学（BD）计算机模拟的酶，涉及辅因子尼古丁腺嘌呤二核苷酸（NAD）。通道假说将测试不仅对这些酶的底物，但也对NAD本身。BD可以探索形成酶-酶复合物的潜力，配体的潜在途径，以及与溶液中分离的酶相比时配体结合的效率。出口将涉及几种酶和不同的物种，看看是否有一个总的趋势，观察整个自然界，包括常见的脊椎动物系统，致病生物和微生物。最初的重点将放在涉及NAD的途径部分，因为酶可以基于已知的晶体结构很好地建模，并且因为可以通过荧光实验跟踪NAD。示例通道模拟包括：甘油醛-3-磷酸脱氢酶（GAPDH）和乳酸脱氢酶（LDH）之间的NAD、果糖-1，6-二磷酸醛缩酶（醛缩酶）和GAPDH之间的甘油醛-3-磷酸（GAP）、磷酸丙糖异构酶（TPI）之间的间隙和GAPDH、醛缩酶、TPI和GAPDH之间的间隙，最后是丙酮酸激酶和LDH之间的丙酮酸。 公共卫生相关性：拟议的工作包括计算机模拟，不仅适用于基础生物医学科学（例如，兔子，人类，大鼠，鸡，斑马鱼，面包酵母），而且适用于传染病生物（例如，恶性疟原虫、锥虫、利什曼原虫、E. coli、M.结核病），试图确定酶-酶相互作用有多普遍，以及跨物种存在何种通道可能性。

DESCRIPTION (provided by applicant): The channeling hypothesis (i.e., that substrates or co-enzymes may move from one active site to another without equilibrating with the bulk medium) is still controversial in glycolysis because glycolytic enzymes are large and have very dynamic interactions that are hard to detect. Channeling has been demonstrated in the tricarboxylic acid cycle where enzyme-enzyme complexes have been crystallized and electrostatic channeling pathways clearly modeled. A variety of experimental methods have provided evidence that channeling may occur in glycolysis including sedimentation, co-pelleting, and electrophoresis. There is also evidence of a metabolon, a functional multienzyme complex, for the glycolytic pathway. Computer modeling simulations can demonstrate potential enzyme-enzyme complexes and follow the trajectories of ligands as they move from one active site to another in such complexes. Herein, exploration of the channeling hypothesis in glycolysis will begin by using Brownian dynamics (BD) computer simulations on the enzymes that involve the co-factor nicotine adenine dinucleotide (NAD). The channeling hypothesis will be tested not only on the substrates of these enzymes, but also on the NAD itself. BD can explore the potential for forming enzyme-enzyme complexes, the potential pathways of ligands, and the efficiency of the ligand binding when compared to isolated enzymes in solution. The exportation will involve several enzymes and different species to see if there is a general trend observed throughout nature, including common vertebrate systems, disease causing organisms, and microorganisms. The initial focus will be on the parts of the pathway involving NAD because the enzymes can be well modeled based on known crystal structures and because it may be possible to follow the NAD through fluorescence experiments. Example channeling simulations include: NAD between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH), glyceraldehyde-3- phosphate (GAP) between fructose-1,6-bisphosphate aldolase (aldolase) and GAPDH, GAP between triose phosphate isomerase (TPI) and GAPDH, GAP among aldolase, TPI and GAPDH, and finally pyruvate between pyruvate kinase and LDH. PUBLIC HEALTH RELEVANCE: The proposed work includes computer simulations useful not only for basic biomedical science (e.g, rabbit, human, rat, chicken, zebra fish, baker's yeast,) but also for infectious disease organisms (e.g., P. falciparum, trypanosomes, Leishmania, E. coli, M. tuberculosis) in an attempt to determine how ubiquitous the enzyme- enzyme interactions are and what kinds of channeling possibilities exist across species.