ADVANCED EPR STUDIES OF B12 DEPENDENT ENZYMES

B12 依赖性酶的高级 EPR 研究

• 批准号: 6039831
• 负责人: GARY J. GERFEN
• 金额: $ 19.24万
• 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 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6039831
• 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 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。 将使用腺苷钴胺素（辅酶 B12）依赖性酶进行研究 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 频率（140 GHz）范围，最终目标是催化机制 决心。特别令人感兴趣的是交换耦合 作为催化中间体产生的 cob(II) 胺-有机自由基对 耳蜗梭菌的谷氨酸变位酶和核糖核苷的循环 来自莱希曼乳杆菌的三磷酸还原酶 (RTPR)。型号钴(ll) 化合物，包括 cob(II)alamin 和 Co(II)-bis(二甲基乙二肟) 也将 进行研究以深入了解光谱特征和 钴胺素的结构，参与交换耦合对 酶。对于谷氨酸变位酶，选择性同位素标记 底物（谷氨酸）（2H、13C 和 15N）可帮助 结构识别。对于 RTPR，与机械抑制剂一起孵育 产生丰富多样的 EPR 光谱。这些自由基种类的识别 将提供对抑制机制甚至催化机制的深入了解。 该项目的总体目标包括：1）结构细节的确定 交换耦合对中酶结合的 cob(II)alamin 物种，通过 赤道和轴向氮超精细和四极参数； 2） 轴配位碱基 I 的远程氮的研究（或者 酶结合的钴（II）丙胺和组氨酸或二甲基苯并咪唑） 交换耦合对； 1 3) 鉴定和结构表征 与酶结合的 cob(II)alamin 偶联的自由基物质； 4）精确 电子-电子交换和零场分裂的测定 （偶极）相互作用来建立和相对之间的距离 两种顺磁物质的方向。这些目标的实现是 有必要全面了解这些催化机制 酶，并且可以深入了解酶在选择性中的作用 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而言，大约为 1 x 10(12) 倍。这个“酶 钴-碳键 j 的活化”是 腺苷钴胺素依赖性催化。