MALATE SYNTHASE - PROOF OF A STEPWISE CLAISEN CONDENSATION USING THE DOUBLE-ISOTOPE FRACTIONATION TEST
MALATE SYNTHASE - PROOF OF A STEPWISE CLAISEN CONDENSATION USING THE DOUBLE-ISOTOPE FRACTIONATION TEST
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DOI：
10.1021/bi00416a020
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发表时间：
1988-08-09
期刊：
影响因子：
2.9
通讯作者：
KNOWLES, JR
中科院分区：
文献类型：
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作者：
CLARK, JD;OKEEFE, SJ;KNOWLES, JR
尽管醛缩酶催化的缩合通过亲核烯醇(酸酯)或烯胺的中间作用逐步进行，但催化克莱森型缩合的那些酶的机制尚不清楚。通过双同位素分级分离方法研究了第二组酶（苹果酸合酶）所遵循的反应途径，以确定该反应是逐步反应还是协同反应。与早期工作一致，氘动力学同位素效应 D(V/K) 为 1.3 .+-。当[2H3]乙酰辅酶A为底物时，发现为0.1。还测量了乙醛酸醛碳上的 13C 同位素效应。为了进行该测定，将苹果酸盐产品（含有C-2处的目标碳）定量转化为具有C-4处的目标碳的新苹果酸盐样品。该材料通过苹果酸酶脱羧，产生适合同位素比质谱分析的二氧化碳。 [1H3]乙酰辅酶A [即 13(V/K)H] 的 13C 同位素效应为 1.0037 .+-。 0.0004。通过使用分子间和分子内氘效应以及 13(V/K)H 的已知值，可以针对三种可能的机制预测当氘代 [2H3]乙酰辅酶 A 为底物 [即 13(V/K)D] 时 13C 同位素效应的值。如果 13(V/K)H 是动力学同位素效应，且反应协同，则 13C 对乙酰辅酶A氘化的效应值将上升至1.011；如果 13(V/K)H 是动力学同位素效应，且反应是逐步进行的，则 13C 效应的值将降至 1.0025；如果 13C 效应是乙醛酸脱水产生的平衡同位素效应，则反应必然是逐步进行的， 13(V/K)D 值为 1.0037，与 13(V/K)H 值不变。实验上，13(V/K)D的值为1.0037.+-。 0.0007，这要求苹果酸合酶遵循逐步路径。因此，很明显，催化类克莱森缩合的酶的两个显着特征，即不存在酶催化的与溶剂的质子交换以及亲核中心的构型反转（这暗示了协同途径），在机械上并不能诊断。
Although aldolase-catalyzed condensations proceed by stepwise mechanisms via the intermediacy of nucleophilic enol(ate)s or enamines, the mechanisms of those enzymes that catalyze Claisen-type condensations are unclear. The reaction pathway followed by an enzyme from this second group, malate synthase, has been studied by the double-isotope fractionation method to determine whether the reaction is stepwise or concerted. In agreement with earlier work, a deuterium kinetic isotope effect D(V/K) of 1.3 .+-. 0.1 has been found when [2H3]acetyl-CoA is the substrate. The 13C isotope effect at the aldehydic carbon of glyoxylate has also been measured. For this determination, the malate product (containing the carbon of interest at C-2) was quantitatively transformed into a new sample of malate having the carbon of interest at C-4. This material was decarboxylated by malic enzyme to produce the appropriate CO2 for isotope ratio mass spectrometric analysis. The 13C isotope effect with [1H3]acetyl-CoA [that is, 13(V/K)H] is 1.0037 .+-. 0.0004. By use of the known values of the intermolecular and intramolecular deuterium effects and of 13(V/K)H, the value of the 13C isotope effect when deuteriated [2H3]acetyl-CoA is the substrate [that is, 13(V/K)D] can be predicted for three possible mechanisms. If 13(V/K)H is a kinetic isotope effect and the reaction is concerted, the value of the 13C effect on deuteriation of acetyl-CoA will rise to 1.011; if 13(V/K)H is a kinetic isotope effect and the reaction is stepwise, the value of the 13C effect will fall to 1.0025; and if the 13C effect is an equilibrium isotope effect deriving from glyoxylate dehydration, the reaction is necessarily stepwise, and the value of 13(V/K)D will be 1.0037, unchanged from that of 13(V/K)H. Experimentally, the value of 13(V/K)D is 1.0037 .+-. 0.0007, which requires that malate synthase follow a stepwise path. It is therefore clear that the two salient characteristics of enzymes that catalyze Claisen-like condensations, namely, the absence of enzyme-catalyzed proton exchange with solvent and the inversion of the configuration at the nucleophilic center, which had been suggestive of a concerted pathway, are not mechanistically diagnostic.