Molecular basis of ATP synthesis machinery from parasite living under microenvironment

2017 Fiscal Year Final Research Report

• Project/Area Number: 16K19114
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Parasitology (including sanitary zoology)
• Research Institution: Nagasaki University
• Principal Investigator: INAOKA Ken Daniel 長崎大学, 熱帯医学・グローバルヘルス研究科, 助教 (10623803)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Keywords: 寄生虫 / ATP合成 / ASCT / 生化学的解析 / 結晶構造解析 / ミトコンドリア様オルガネラ

Outline of Final Research Achievements

Trypanosoma brucei produces acetate (Ace) in mitochondria by two different enzymes: acetyl-CoA (ACoA) hydrolase or acetate:succinate CoA-transferase (ASCT). Under physiological condition, ASCT transfers the CoA from ACoA to succinate (Suc) to form Ace and succinyl-CoA (SCoA), which is consequently converted back to Suc by SCoA synthase (SCS) with net production of 1 mol of ATP per mol of ACoA in a process named ASCT/SCS cycle. So far, no biochemical information for ASCT family of CoA-transferase is reported. In this project, an over-expression system and purification of active TbASCT together with a novel sensitive method to measure ASCT activity have been developed. Purified TbASCT obeys Michaelis-Menten kinetics with Kcat of 202 ATP per second and show high affinity for ACoA (Km = 47 μM) than succinate (9.86 mM). A method for in-gel ASCT activity staining was developed showing evidence that TbASCT is active as homotetramer which was consistent to the crystal structure.

Free Research Field

寄生虫学(含衛生動物学)