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）：人类的疾病是由