Clark J D, O'Keefe S J, Knowles J R
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
Biochemistry. 1988 Aug 9;27(16):5961-71. doi: 10.1021/bi00416a020.
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.
尽管醛缩酶催化的缩合反应通过亲核烯醇（盐）或烯胺中间体以逐步机制进行，但催化克莱森型缩合反应的那些酶的机制尚不清楚。已通过双同位素分馏法研究了第二组中的一种酶——苹果酸合酶所遵循的反应途径，以确定该反应是逐步进行还是协同进行。与早期工作一致，当[2H3]乙酰辅酶A作为底物时，发现氘动力学同位素效应D(V/K)为1.3±0.1。还测量了乙醛酸醛基碳上的13C同位素效应。为了进行该测定，将苹果酸产物（在C-2处含有感兴趣的碳）定量转化为在C-4处含有感兴趣的碳的苹果酸新样品。该物质通过苹果酸酶脱羧以产生用于同位素比率质谱分析的合适CO2。[1H3]乙酰辅酶A的13C同位素效应[即13(V/K)H]为1.0037±0.0004。利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作为底物时13C同位素效应的值[即13(V/K)D]。如果13(V/K)H是动力学同位素效应且反应是协同的，乙酰辅酶A氘代时13C效应的值将升至1.011；如果13(V/K)H是动力学同位素效应且反应是逐步的，13C效应的值将降至1.0025；如果13C效应是源自乙醛酸脱水的平衡同位素效应，则反应必然是逐步的，且13(V/K)D的值将为1.0037，与13(V/K)H的值相同。实验上，13(V/K)D的值为1.0037±0.0007，这表明苹果酸合酶遵循逐步途径。因此很明显，催化类克莱森缩合反应的酶的两个显著特征，即不存在酶催化的与溶剂的质子交换以及亲核中心构型的翻转，这曾暗示协同途径，但在机制上并无诊断意义。
