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（根据摘要改编）：人类疾病是由 维生素B12的缺乏或吸收不良包括高脑结晶质血症， 大型细胞贫血，恶性贫血和甲基丙二酸酯血症。 将使用依赖性酶（辅酶B12）依赖性酶进行研究 多频EPR，ESEEM（电子自旋回波信封调制）和endor （电子核双共振）低频（4-18 GHz）和高 具有催化机制的最终目标的频率（140 GHz）制度 决心。特别感兴趣的是交换耦合 催化剂中的中间体产生的COB（LL）丙氨酸有机自由基对 谷氨酸突变酶的循环来自梭状芽胞杆菌和核糖核苷的循环 来自乳杆菌的三磷酸还原酶（RTPR）。模型钴（LL） 包括COB（LL）Alamin和CO（LL）-BIS（二甲基差异）的化合物也将 进行研究以洞悉光谱特征和 钴胺素的结构，该结构涉及在交换耦合对中 酶。对于谷氨酸突变酶的情况，有选择性地标记 底物（谷氨酸）可用（2H，13C和15N），以帮助 结构识别。对于RTPR，与机械抑制剂一起孵育 产生各种EPR光谱。这些激进物种的识别 将提供有关抑制机制以及可能催化的洞察力。 该项目的总体目标包括：1）确定结构细节 通过交换耦合对中酶结合的蛋白棒（LL）alamin物种 赤道和轴向氮超细和四极参数； 2） 研究轴向协调的碱基I的远程氮（要么 酶结合的COB（LL）alamin和 交换耦合对； 1 3）识别和结构表征 与结合酶结合的蛋白酶（LL）alamin耦合的激进物种； 4）精确 电子电子交换和零场拆分的确定 （偶极）相互作用以建立和相对之间的距离 两种顺磁性物种的方向。这些目标的实现是 必须完整地了解这些催化机制 酶，可以深入了解酶在有选择的 通过增加腺苷泡蛋白碳质粘结键的均质反应性的提高 相对于游离的腺苷胆质，约为1 x 10（12）。这个酶 激活“钴 - 碳键J是对 腺苷核蛋白依赖性催化。