Acid-base chemical mechanism of homocitrate synthase from Saccharomyces cerevisiae.
Acid-base chemical mechanism of homocitrate synthase from Saccharomyces cerevisiae.
复制标题
酿酒酵母高柠檬酸合酶的酸碱化学机制。
DOI：
10.1021/bi060889h
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Cook,PaulF
中科院分区：
文献类型：
--
作者：
Qian,Jinghua;West,AnnH;Cook,PaulF
高柠檬酸合酶（乙酰辅酶A：2-酮戊二酸C-转移酶; E. C. 2.3.3.14）催化AcCoA和α-酮戊二酸缩合生成高柠檬酸盐和CoA。该酶被发现是一种含锌的金属酶，使用电感耦合等离子体质谱。使用α-酮戊二酸的死端类似物获得α-酮戊二酸结合位点的拓扑信息。α-酮戊二酸的α-羧酸和α-氧代基团是与活性位点Zn配位的最佳结合所必需的。α-酮戊二酸的α-羧酸酯、α-氧代和γ-羧酸酯的最佳位置可能由2，4-二羧酸吡啶的2-羧酸酯、吡啶氮和4-羧酸酯的空间位置模拟。的pH依赖性的动力学参数进行了测定，以获得高柠檬酸合酶的化学机制的信息。V曲线呈钟形，斜率为1和−1，pKa值为6.7和8.0，而V/KAcCoA曲线的酸性侧斜率为2，平均pKa值为6.6，碱性侧斜率为−2，平均pKa值为8.2。V/Kα-KgpH−速率曲线显示酸性侧的单个pKa为6.9，碱性侧为2，平均值为7.8。与α-酮戊二酸相比，乙醛酸是一种竞争性抑制剂，其Ki的pH依赖性使一个基团的pKa为7.1，需要质子化以实现最佳结合。数据表明该酶的化学机制为α-酮戊二酸首先通过其α-羧酸和α-氧代基团与活性位点Zn结合，然后是乙酰辅酶A。然后，一般的碱从乙酰辅酶A的甲基接受一个质子，一般的酸使α-酮戊二酸的羰基质子化，形成高柠檬酰辅酶A。然后，在高柠檬酰-CoA的水解中，一般的酸在Zn-OH 2的去质子化中充当碱，得到高柠檬酸盐和CoA。高柠檬酸合酶的溶剂氘动力学同位素效应为1，而使用氘代乙酰辅酶A观察到小的pH独立的主要动力学氘同位素效应（Δ 1.3）。数据表明限速缩合以形成高柠檬基-CoA的醇盐，然后水解以得到产物。
Homocitrate synthase (acetyl-coenzyme A:2-ketoglutarate C-transferase; E.C. 2.3.3.14) catalyzes the condensation of AcCoA and α-ketoglutarate to give homocitrate and CoA. The enzyme was found to be a Zn-containing metalloenzyme using inductively coupled plasma mass spectrometry. Dead-end analogues of α-ketoglutarate were used to obtain information on the topography of the α-ketoglutarate binding site. The α-carboxylate and α-oxo groups of α-ketoglutarate are required for optimum binding to coordinate to the active site Zn. Optimum positioning of the α-carboxylate, α-oxo, and γ-carboxylate of α-ketoglutarate is likely mimicked by the location in space of the 2-carboxylate, pyridine nitrogen, and 4 carboxylate of pyridine 2,4-dicarboxylate. The pH dependence of the kinetic parameters was determined to obtain information on the chemical mechanism of homocitrate synthase. TheVprofile is bell shaped with slopes of 1 and −1, giving pKavalues of 6.7 and 8.0, whileV/KAcCoAexhibits a slope of 2 on the acidic side with an average pKavalue of 6.6 and a slope of −2 on basic side of the profile with an average pKavalue of 8.2. TheV/Kα-KgpH−rate profile exhibits a single pKaof 6.9 on the acidic side and two on the basic side with an average value of 7.8. The pH dependence of theKifor glyoxylate, a competitive inhibitor vs α-ketoglutarate, gives a pKaof 7.1 for a group, required to be protonated for optimum binding. Data suggest a chemical mechanism for the enzyme in which α-ketoglutarate first binds to the active site Zn via its α-carboxylate and α-oxo groups, followed by acetyl-CoA. A general base then accepts a proton from the methyl of acetyl-CoA, and a general acid protonates the carbonyl of α-ketoglutarate in the formation of homocitryl-CoA. The general acid then acts as a base in deprotonating Zn-OH2in the hydrolysis of homocitryl-CoA to give homocitrate and CoA. A solvent deuterium kinetic isotope effect of 1 is measured for homocitrate synthase, while a small pH-independent primary kinetic deuterium isotope effect (∼1.3) is observed using deuterioacetyl-CoA. Data suggest rate-limiting condensation to form the alkoxide of homocitryl-CoA, followed by hydrolysis to give products.