ADVANCED EPR STUDIES OF B12 DEPENDENT ENZYMES

B12 依赖性酶的高级 EPR 研究

• 批准号: 6498827
• 负责人: GARY J. GERFEN
• 金额: $ 20.16万
• 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/6498827
• 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。 腺苷钴胺(辅酶B12)依赖的酶将使用 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 频率(140 GHz)制度，最终目标是催化机制 决心。特别令人感兴趣的将是交易所耦合 钴(Ⅱ)丙胺-有机自由基对在催化反应中的中间体生成 耳壳梭菌谷氨酸变位酶与核糖核苷的循环 莱氏乳杆菌的三磷酸还原酶(RTPR)。模型钴(11) 化合物，包括钴(11)丙胺和钴(11)-二(丁二酮肟)也将 研究以提供对光谱特征的洞察和 参与交换偶联对的钴胺的结构 酵素。对于谷氨酸变位酶的情况，选择性地同位素标记 底物(谷氨酸盐)可用(2H、13C和15N)来帮助 结构识别。对于RTPR，与机械抑制剂孵育 产生丰富的EPR谱。这些自由基物种的鉴定 将提供对抑制的机制的洞察，甚至是催化的机制。 本项目的总体目标包括：1)确定结构细节 交换偶联对中的酶结合的CoB(11)丙胺物种，通过 赤道和轴向氮超精细和四极参数；2) 轴向配位碱基I(任一)的远程氮的研究 组氨酸或二甲基苯并咪唑)在酶结合的CoB(11)丙氨酸和 交换偶联对.1)鉴定和结构表征 与酶结合的CoB(11)丙氨酸偶联的自由基物种；4)精确 电子-电子交换和零场分裂的测定 (偶极)相互作用以确定和之间的距离 这两个顺磁物种的取向。这些目标的实现是 有必要完整地描述这些化合物的催化机理 酶，并可能深入了解酶在选择性地 通过以下方法提高腺苷钴胺碳-钴键的均裂反应活性 相对于游离腺苷钴胺的约1×10(12)倍。这种“酶” 钴-碳键j的活化是 腺苷钴胺依赖催化。