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首页> 外文期刊>Biochemistry >How fumarase recycles after the malate -> fumarate reaction. Insights into the reaction mechanism
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How fumarase recycles after the malate -> fumarate reaction. Insights into the reaction mechanism

机译：苹果酸->富马酸酯反应后，富马酸酯如何循环利用。洞悉反应机制

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Recycling of yeast fumarase to permit repetition of its reaction chemistry requires two proton transfers and two conformational changes, in pathways that are different in detail but thematically similar in the two directions. In the malate --> fumarate direction, simple anions such as acetate accelerate the fumarate-off step producing E-H(f), a fumarate-specific isoform that retains the C3R-proton of malate, Fumarate specificity is shown with S-2,3-dicarboxyaziridine, which is competitive vs fumarate and noncompetitive with malate as substrate. The steady-state level of E-H(f), based on K-ii (S-2,3-dicarboxyaziridine), is increased by D2O and decreased by imidazole acting as a general acid for conversion of E-H(f) to E-H(f)(H). E-H(f)(H) is fumarate-specific as shown by the inhibition pattern with ClO4-. The pK(a) of this step is similar to 7.25 based on the pH dependence of K-ii (ClO4-). A conformational change occurs next as shown by high sensitivity of k(cat) but not k(cat)/K-m, to the microviscosogen, glycerol, and change to a nonspecific isoform, E-H(mf)(H), probably the same species formed in the fumarate --> malate direction from malate-specific intermediates by a different conformational change, Malate enters the cycle by reaction with E-H(mf)(H) and returns to E-(m)(H).malate after a second conformational change. When fumarate-off is slow, as in low anion medium, malate itself becomes an activator of malate --> fumarate, This effect occurs with changes in inhibition patterns suggestive of the bypass of the slow E-(f) --> E-(mf) conversion in favor of direct formation of E-(mf) when free fumarate is formed. 3-Nitro-2-hydroxypropionate, a strong inhibitor of fumarase [Porter, D. J. T., and Bright, H. J. (1980) J. Biol. Chem. 255, 4772-4780] in its carbanion form, is competitive with both malate and fumarate, Therefore, 3-nitro-2-hydroxypropionic acid interacts with E-H(mf)(H) and not with E-(m) or E-(f) isoforms. Occurrence of two different conformational changes in the recycling process suggests that the reaction chemistry employs a two-step mechanism, The specificity of inhibition for E-H(mf)(H) is consistent with the expected intermediate of a carbanion mechanism, EH carbanion(-). The proton transfers and conformational changes of recycling occur in the same sequence that is expected for this reaction chemistry. Several examples of ligand-activated conformational changes are reported. [References: 17]
机译：再循环酵母富马酶以重复其反应化学过程，需要两个质子转移和两个构象变化，其路径在细节上不同，但在两个方向上在主题上相似。在苹果酸->富马酸酯方向上，简单的阴离子（如乙酸盐）加速了富马酸酯的分离步骤，产生了EH（f），这是一种富马酸酯特异性同工型，保留了苹果酸的C3R质子，富马酸酯的特异性如S-2,3所示。 -二羧基氮丙啶，与富马酸酯竞争，并且以苹果酸为底物不竞争。基于K-ii（S-2,3-二羧基氮丙啶）的EH（f）的稳态水平通过D2O升高，而咪唑作为将EH（f）转化为EH（f ）（H）。 E-H（f）（H）具有富马酸酯特异性，如ClO4-的抑制模式所示。基于K-ii（ClO4-）的pH依赖性，此步骤的pK（a）类似于7.25。接下来发生构象变化，这是由k（cat）/ km对微粘菌素甘油的高敏感性所显示的，而不是k（cat）/ Km，并变为非特异性同种型EH（mf）（H），可能形成了相同的物种通过不同的构象变化，从富马酸盐->苹果酸特定中间体的苹果酸方向开始，苹果酸通过与EH（mf）（H）反应进入循环，并在第二构象后返回E-（m）（H）。更改。当富马酸酯的释放缓慢时，例如在低阴离子培养基中，苹果酸本身会成为苹果酸的活化剂->富马酸酯，这种抑制作用会随着抑制模式的改变而发生，提示绕开了慢E-（f）-> E- （mf）转化有利于在形成游离富马酸酯时直接形成E-（mf）。 3-硝基-2-羟基丙酸酯，富马酸酯的强抑制剂[Porter，D.J.T。和Bright，H.J。（1980）J。化学[255，4772-4780]的碳负离子形式与苹果酸和富马酸酯竞争，因此3-硝基-2-羟基丙酸与EH（mf）（H）相互作用，而不与E-（m）或E-（ f）同工型。在回收过程中发生两种不同的构象变化表明，反应化学采用了两步机制，对EH（mf）（H）的抑制特异性与预期的碳负离子中间产物EH碳负离子（-）一致。 。质子转移和循环构象变化以与该反应化学预期相同的顺序发生。报告了配体激活的构象变化的几个例子。 [参考：17]

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