Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs.
Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs.
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DOI：
10.1016/j.bbrc.2013.10.066
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发表时间：
2013-11-15
影响因子：
3.1
通讯作者：
St Maurice, Martin
中科院分区：
文献类型：
--
作者：
Lietzan, Adam D.;St Maurice, Martin
丙酮酸羧化酶（PC）是一种生物素依赖性酶，催化丙酮酸盐的MgATP和碳酸氢盐依赖性羧化为草酰乙酸，这是中枢代谢中的重要回补反应。PC的羧基转移酶（CT）结构域催化羧基从羧基生物素转移到接受底物丙酮酸。已经假设反应性烯醇丙酮酸中间体通过与CT结构域活性位点中的金属离子的双齿相互作用而稳定。虽然双齿配体通常在通过烯醇丙酮酸中间体进行的酶催化反应中观察到，但在PC的CT结构域中尚未观察到双齿相互作用。在这里，我们报告了三个X-射线晶体结构的根瘤菌etli PC CT域结合抑制剂草酸，3-羟基丙酮酸，和3-溴丙酮酸。草酸盐是烯醇丙酮酸中间体的立体电子模拟物，不直接与金属离子相互作用。相反，草酸盐被埋在由几个带正电荷的氨基酸残基和金属离子形成的口袋中。此外，3-羟基丙酮酸和3-溴丙酮酸，反应产物草酰乙酸的类似物，以相同的方式结合到草酸盐，这表明底物在整个催化过程中保持其在活性位点的取向。总之，这些结构表明PC催化反应中的底物、产物和中间体并不像先前假设的那样在活性位点中取向。与活性中心金属不存在双齿相互作用似乎是作用于α-酮酸底物的一小类生物素依赖性酶的独特机制特征。
Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates.
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生物素羧基载体结构域与丙酮酸羧化酶中的生物素羧化酶结构域之间的相互作用。
影响因子：
2.9
作者：
Lietzan, Adam D.;Menefee, Ann L.;Zeczycki, Tonya N.;Kumar, Sudhanshu;Attwood, Paul V.;Wallace, John C.;Cleland, W. Wallace;St Maurice, Martin
通讯作者：
St Maurice, Martin
影响因子：
2.8
作者：
CHARLES, AM;WILLER, DW
通讯作者：
WILLER, DW
影响因子：
2.6
作者：
CHARLES, AM;WILLER, DW;SCHARER, JM
通讯作者：
SCHARER, JM
影响因子：
16.8
作者：
Xiang, Song;Tong, Liang
通讯作者：
Tong, Liang
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K