Structural, Association, and Kinetic Properties of Dehydrogenases

自1940年以来，具有相反手性特异性的脱氢酶（DeHs）偶联反应的异常动力学已经得到了很好的记录。对这种异常现象的唯一简单解释是，供体酶与NADH的复合物（El NADH）可以直接将NADH转移到受体酶E2上，从而使NADH与整体水相不显着平衡。也就是说，NADH是E2的活性底物。这种“通灵”的潜在代谢后果比通常认识到的要大得多。例如，1)通道允许酶反应独立于体相底物浓度进行，2)通道将代谢物从竞争反应中分离出来，3)酶结合底物避免了细胞溶剂化能力不足，4)与酶结合的底物的脱溶状态可能是酶率提高的主要因素，5)通道允许酶根据最近扩展的代谢控制理论进行更大程度的控制。6)通道系统受影响酶结合的调节剂的调节。大多数这些后果是由代谢产物的微区隔造成的，这是该机制固有的。然而，在DeHs中NADH通道的一个令人困惑的方面是在体外观察El NADH E2复合物的困难。Peticolas等人似乎已经解决了这个问题，他们证明了这两种酶的结合主要取决于NADH的浓度。他们的实验仅限于a-甘油- p （aGPD）和乳酸（LDH） DeH酶对（加上两个a手性特异性DeH的阴性对照），两种NADH浓度和单一实验方法。由于这一过程在代谢中的潜在意义，我们提出以下重叠目标。1)表征脱氢酶结合的NADH调节：a)更明确地建立LDH-aGPD系统的性质，b)测试NADH调节在其他酶对中的普遍性。2)将这些酶结合与不同酶对之间底物通道的效率联系起来；3)将这些结合和通道特性与供体酶和受体酶的分子结构联系起来。酶关联将通过荧光极化、琼脂糖凝胶电泳、Hummel-Dreyer大小排斥色谱、分析性超离心和差热滴定法进行研究。此外，E1与E2的结合仅发生在E2催化反应的稳态期间的可能性将通过计算机模拟进行验证。NADH通道将通过“酶缓冲法”和代谢控制理论和实验来表征。这些功能特性将与酶结构相关联，使用分子图和相关程序来推断具有已知晶体结构的近15种DeH酶的共同决定因素。事实上，只有A-B对DeHs通道NADH为我们提供了实现这些目标的绝佳机会。在一项高度相关的研究中，代谢物的原位通道也将使用渗透性PC12细胞进行测试。酶负责所有的基本代谢反应以及其他反应，例如，细胞遗传信息的复制和翻译以及细胞的信号传导过程。经典的细胞模型假设主要代谢途径的大多数酶（以及它们的代谢产物）是随机分布在它们的细胞区室内的。此外，我们对代谢调节的理解在很大程度上依赖于这种简化的假设。然而，越来越多的有力证据表明，这些酶中的许多都与代谢序列中的下一个酶有关。这允许代谢产物从一种酶直接转移到另一种酶，而不会在细胞内随机分布。如果发生这种情况，代谢特性将与我们目前认为存在的代谢特性显著不同。因此，我们计划在一类重要的酶中测试这些酶关联的存在，并确定这些关联对这些酶催化的代谢反应的影响。由于技术原因，这些酶的结合在细胞外比在细胞内要小得多。因此，需要特殊的方法来检测细胞外的关联，并且也将尝试在原位测量通道。此外，许多这些酶的分子结构现在已经从x射线研究中得知。因此，我们将把这些分子结构与酶的关联和代谢特性联系起来。* * * ? ?

Abstract 9513613 Anomalous kinetics for the coupled reactions of dehydrogenases (DeHs) of opposite chiral specificity have been well documented since 1940. The only simple explanation of the anomalies is that the complex of donor enzyme with NADH (El NADH) can directly transfer NADH to an acceptor enzyme E2 so that NADH doesn't significantly equilibrate with the bulk aqueous phase. That is: NADH is a competent substrate for E2. The potential metabolic consequences of this "channeling" are far greater than commonly realized. For example, 1) channeling permits enzyme reactions to proceed independently of bulk phase substrate concentrations, 2) channeling isolates metabolites from competing reactions, 3) enzyme bound substrates avoid the inadequate solvation capacity of the cell, 4) the desolvated state of substrates bound to enzymes may be a major factor in enzyme rate enhancement, 5) channeling permits larger degree of control by the enzymes according to recently extended metabolic control theory, and 6) channeling systems are subject to regulation by modulators that affect enzyme associations. Most of these consequences result from the microcompartmentation of metabolites that is inherent in this mechanism. A puzzling aspect of NADH channeling among the DeHs, however, was the difficulty in observing the El NADH E2 complex in vitro. Peticolas et al. appear to have resolved this problem by demonstrating that association of the two enzymes depends critically on the NADH concentration. Their experiments were limited to the a-glycerol-P (aGPD) & lactate (LDH) DeH enzyme pair (plus a negative control with two A chiral specific DeHs), two NADH concentrations, and a single experimental method. Because of the potential significance of this process in metabolism, we propose the following overlapping objectives. 1) Characterize the NADH modulation of the association of dehydrogenases to: a) more definitively establish the properties of the LDH-aGPD system, and b) test the generality of this NADH modulat ion among other enzyme pairs. 2) Correlate these enzyme associations with the efficiencies of substrate channeling among different enzyme pairs, and 3) Correlate these association and channeling properties with the molecular structures of the donor and acceptor enzymes. Enzyme associations will be studied by fluorescence polarization, agarose gel electrophoresis, Hummel-Dreyer size exclusion chromatography, analytical ultracentrifugation, and differential thermal titration methods. In addition, the possibility that the association of E1 with E2 occurs only during the steady-state of the E2 catalyzed reaction will be tested by computer simulations. NADH channeling will be characterized by the "enzyme buffering method" and metabolic control theory and experiments. These functional properties will be correlated with enzyme structures using molecular graphics and related programs to deduce the common determinants among the nearly 15 DeH enzymes with known crystal structures. The fact that only A-B pairs of DeHs channel NADH provides us an excellent opportunity to achieve these objectives. In a highly related study, the channeling of metabolites in situ will also be tested using permeabilized PC12 cells. %%% Enzymes are responsible for all basic metabolic reactions as well as other reactions, e.g., those replicating and translating the genetic information of cells and the signaling processes of cells. The classical model of the cell assumes that most of the enzymes (and consequently also their metabolic products) of the major metabolic pathways are randomly distributed within their cell compartments. Furthermore, our understanding of the regulation of metabolism is significantly dependent on this simplifying assumption. Very strong evidence has accumulated, however, that instead, many of these enzymes are associated with the next enzyme in the metabolic sequence. This permits the direct transfer of metabolic products from one enzyme to the next without their random distribution within the cell comp artment. If this occurs, metabolic properties will be significantly different from those that we currently assume to exist. Consequently, we plan to test for the presence of these enzyme associations among an important class of enzymes and to also determine the consequences of these associations on the metabolic reactions that these enzymes catalyze. For technical reasons, these enzyme associations are much smaller outside the cell than within. Thus special methods are required to detect the associations outside the cell and measurements of channeling in situ will also be attempted. In addition, the molecular structures of many of these enzymes are now known from x-ray studies. Thus we shall correlate these molecular structures with the enzyme association and metabolic properties. *** ??