STRUCTURAL STUDIES OF SUCCINYL COA SYNTHETASE & COA TRANSFERASE

琥珀酰辅酶A合成酶的结构研究

• 批准号: 6483477
• 负责人: MICHAEL JAMES
• 金额: $ 12.06万
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-15 至 2002-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6483477
• 关键词: biomedical resource; structural biology

Succinyl-CoA synthetase (SCS) catalyzes the substrate-level phosphorylation step of the citric acid cycle. In ketone body metabolism, SCS generates succinyl-CoA from succinate, CoA and ATP; and CoA transferase (CoAT) uses this succinyl-CoA in the activation of the ketone body acetoacetate, producing succinate and acetoacetyl-CoA. We have determined the structure of E. coli SCS using 23 crystals. We intend to collect data to the diffraction limit of these crystals using one crystal and cryotemperatures. These crystals grow in the presence of CoA and the structure determination demonstrated the CoA-binding site. The binding site for the nucleotide was detem-iined using data from crystals soaked with ADP, proving that the histidine residue phosphorylated in the reaction must swing -30 A between the binding site for CoA and the binding site for the nucleotide. To interpret the catalytic mechanism, the binding site of succinate or the succinyl moiety must be known. Crystals of pig heart SCS grow in the absence of CoA and we have successfully soaked them with CoA to determine the structure of the complex. We propose collecting data from crystals soaked with a non-hydrolyzable analogue of succinyl-CoA to determine the succinyl binding site. The structure of CoA transferase is yet to be determined because attempts at soaking the monoclinic crystals with heavy atoms have led to noniso-morphism. Data collection was on BioCARS Station 14 BM-C.

琥珀酰辅酶A合成酶(SCS)催化底物水平 柠檬酸循环的磷酸化步骤。在酮体中 代谢过程中，SCS由琥珀酸、辅酶A和三磷酸腺苷合成琥珀酰辅酶A； CoA转移酶(CoAT)使用这种琥珀酰辅酶A来激活 酮体乙酰乙酸酯，产生丁二酸酯和乙酰乙酰-辅酶A。 我们已经用23个晶体确定了大肠杆菌SCS的结构。我们 打算收集数据以达到这些晶体的衍射极限 使用一种晶体和低温。这些晶体生长在 CoA的存在和结构测定证明了 CoA结合位点。确定了核苷酸的结合部位 使用来自浸泡ADP的晶体的数据，证明组氨酸 反应中磷酸化的残留物必须在-30A之间摆动 CoA的结合部位和核苷酸的结合部位。至 解释催化机理，琥珀酸根的结合部位 琥珀酸基部分必须是已知的。猪心干细胞中生长的晶体 没有CoA，我们已经成功地用CoA浸泡了他们 确定复合体的结构。我们建议收集数据 用琥珀酰辅酶A的非水解性类似物浸泡的晶体 测定琥珀酸基结合部位。CoA的结构 转移酶尚未确定，因为试图浸泡 含有重原子的单斜晶系导致了不同构型。 数据采集在BioCARS 14BM-C站进行。