ADVANCED EPR STUDIES OF B12 DEPENDENT ENZYMES

B12 依赖性酶的高级 EPR 研究

• 批准号: 6628902
• 负责人: GARY J. GERFEN
• 金额: $ 20.73万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6628902
• 关键词: benzimidazole analog; carbon; chemical structure function; cobalamin; cobalt; cobamide; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme mechanism; enzyme structure; glutamates; histidine; isomerase; nitrogen; nucleoside triphosphate; oxidoreductase; ribonucleosides

DESCRIPTION (Adapted from abstract): Diseases in humans that are caused by deficiencies or malabsorption of vitamin B12 include hyperhomocysteinemia, megaloblastic anemia, pernicious anemia and methylmalonic acidemia. Adenosylcobalamin (Coenzyme B12)-dependent enzymes will be studied using multifrequency EPR, ESEEM (electron spin echo envelope modulation), and ENDOR (electron nuclear double resonance) in the low frequency (4-18 GHz) and high frequency (140 GHz) regimes with the ultimate goal of catalytic mechanism determination. Of particular interest will be the exchange-coupled cob(ll)alamin-organic radical pairs generated as intermediates in the catalytic cycles of glutamate mutase from Clostridium cochlearium and ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannIi. Model cobalt(ll) compounds, including cob(ll)alamin and Co(ll)-bis(dimethylglyoximes) will also be studied to provide insight into the spectroscopic characteristics and structure of the cobalamin which is involved in exchange-coupled pairs in the enzymes. For the case of glutamate mutase, selectively isotopically labeled substrates (glutamates) are available (2H, 13C and 15N) to aid in the structural identification. For RTPR, incubation with mechanistic inhibitors produces a rich variety of EPR spectra. Identification of these radical species will provide insight into the mechanism of inhibition and perhaps of catalysis. Overall goals of this project include: 1) determination of structural details of the enzyme-bound cob(ll)alamin species in the exchange-coupled pair, through equatorial and axial nitrogen hyperfine and quadrupole parameters; 2) investigation of the remote nitrogen of the axially coordinated base I (either histidine or dimethylbenzimidazole) in both the enzyme-bound cob(ll)alamin and exchange-coupled pair; 1 3) identification and structural characterization of the radical species coupled to the enzyme-bound cob(ll)alamin; 4) precise determination of the electron-electron exchange and zero field splitting (dipolar) interaction to establish the distance between and relative orientation of the two paramagnetic species. Achievement of these aims is necessary to give a complete picture of the catalytic mechanisms of these enzymes, and may give insight into the role of the enzyme in selectively increasing homolytic reactivity of the adenosylcobalamin carbon-cobalt bond by a factor of about 1 x 10(12) relative to free adenosylcobalamin. This "enzyme activation" of the cobalt-carbon bond j is a requirement for adenosylcobalamin-dependent catalysis.

描述（改编自摘要）：由以下原因引起的人类疾病 维生素B12的缺乏或吸收不良包括高同型半胱氨酸血症， 巨幼细胞性贫血、恶性贫血和甲基丙二酸血症。 腺苷钴胺素（辅酶B12）依赖性酶将使用 多频EPR、ESEEM（电子自旋回波包络调制）和ENDOR （电子核双共振）在低频（4-18 GHz）和高 频率（140 GHz）制度与催化机制的最终目标 保持战略定力特别感兴趣的将是交换耦合 Cob（II）丙氨酸-有机自由基对作为催化反应中的中间体产生， 耳蜗梭菌谷氨酸代谢循环与核糖核苷 来自莱氏乳杆菌的三磷酸还原酶（RTPR）。模型钴（II） 包括钴（II）丙氨酸和钴（II）-双（二甲基乙二醛）的化合物也将 进行研究，以深入了解光谱特性， 钴胺素的结构，这是参与交换耦合对在 内切酶对于谷氨酸盐的情况，选择性同位素标记 底物（谷氨酸）可用（2 H，13 C和15 N），以帮助 结构识别对于RTPR，与机制抑制剂孵育 产生丰富的EPR谱。识别这些自由基物质 将提供对抑制机制的深入了解，也许还有催化机制。 本项目的总体目标包括：1）确定结构细节 在交换偶联对中的酶结合的cob（ll）alamin物质，通过 赤道和轴向氮超精细和四极参数; 2） 轴向配位碱基I的远程氮的研究（或者 组氨酸或二甲基苯并咪唑）在酶结合的辅基（II）丙氨和 交换耦合对; 1 3）识别和结构表征 自由基物质与酶结合的辅酶B（II）丙氨酸; 4）精确 电子-电子交换和零场分裂的测定 （偶极）相互作用，以建立和相对 两种顺磁性物质的取向。实现这些目标是 有必要给出一个完整的图片的催化机制，这些 酶，并可能使洞察酶的作用，选择性地 增加腺苷钴胺素碳-钴键的均裂反应性， 相对于游离腺苷钴胺素约为1 × 10（12）倍。这种“酶 钴-碳键j的“活化”是 腺苷钴胺素依赖性催化作用。