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Acetate: succinate CoA transferase (ASCT), together with succinyl\u2010CoA synthase (SCS), forms the ASCT\/SCS cycle that fuels ATP production and generates acetate, a central metabolic intermediate essential for mitochondrial pathways in these parasites. Although\n Trypanosoma brucei<\/jats:italic>\n ASCT (TbASCT) shares 52% amino acid identity with mammalian succinyl\u2010CoA:3\u2010ketoacid CoA transferase (mSCOT), the latter catalyzes a rate\u2010limiting step of ketone body catabolism. Because ASCT and SCOT perform distinct reactions, understanding their mechanistic divergence is crucial for identifying parasite\u2010specific vulnerabilities and advancing selective drug discovery. Here, we report crystal structures of TbASCT bound to all substrates and products, revealing the molecular basis of substrate recognition and catalysis. In solution, TbASCT and mSCOT are homotetrameric and homodimeric, respectively. Despite similar monomer fold, the substrate\u2010binding sites and catalytic mechanisms by which these two enzymes mediate different reactions remain unknown. Site\u2010directed mutagenesis demonstrated that residues Arg162, Leu377, and Asp62 govern tetramer assembly, CoA binding, and acquisition of SCOT activity, respectively. Mutation of Leu377 abolished ASCT activity, while Arg162 mutant produced ASCT\u2010active dimers with a 10\u2010fold increase in SCOT\/ASCT activity ratio. Notably, Asp62 mutants exhibited more than 4000\u2010fold increase in this ratio, representing gain\u2010of\u2010function SCOT activity, a reaction absent in TbASCT. These mechanistic insights define the structural determinants that separate ASCT from SCOT function and illuminate opportunities to selectively inhibit ASCT without disrupting host SCOT, thereby informing the development of trypanosomatid\u2010targeted therapeutics.\n <\/jats:p>","DOI":"10.1002\/pro.70463","type":"journal-article","created":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T12:41:17Z","timestamp":1768912877000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Modulation of succinyl\u2010\n CoA<\/scp>\n :3\u2010ketoacid\n CoA<\/scp>\n transferase activity by a single amino acid residue in acetate:succinate\n CoA<\/scp>\n transferase from\n Trypanosoma brucei<\/i>\n , the causative agent of African sleeping sickness"],"prefix":"10.1002","volume":"35","author":[{"given":"Kota","family":"Mochizuki","sequence":"first","affiliation":[{"name":"Department of Immunogenetics, Institute of Tropical Medicine 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