biostudies-literatureMeng YSouth Carolina Experiment StationNIHNIH HHSNIGMS NIH HHS1643https://www.ebi.ac.uk/biostudies/studies/S-EPMC10455477biostudies-literatureUnknown13(8)Short- and medium-chain acyl-CoA synthetases catalyze similar two-step reactions in which acyl substrate and ATP bind to form an enzyme-bound acyl-adenylate, then CoA binds for formation of the acyl-CoA product. We investigated the roles of active site residues in CoA binding in acetyl-CoA synthetase (Acs) and a medium-chain acyl-CoA synthetase (Macs) that uses 2-methylbutyryl-CoA. Three highly conserved residues, Arg¹⁹³, Arg⁵²⁸, and Arg⁵⁸⁶ of <i>Methanothermobacter thermautotrophicus</i> Acs (Acs_Mt), are predicted to form important interactions with the 5'- and 3'-phosphate groups of CoA. Kinetic characterization of Acs_Mt variants altered at each of these positions indicates these Arg residues play a critical role in CoA binding and cLife (Basel, Switzerland)The Roles of Coenzyme A Binding Pocket Residues in Short and Medium Chain Acyl-CoA Synthetases.PMC10455477R15 GM069374SC-1700198GM69374-01A1Ahmed OMeng YSmith KIngram-Smith CfalseThe Roles of Coenzyme A Binding Pocket Residues in Short and Medium Chain Acyl-CoA Synthetases.Short- and medium-chain acyl-CoA synthetases catalyze similar two-step reactions in which acyl substrate and ATP bind to form an enzyme-bound acyl-adenylate, then CoA binds for formation of the acyl-CoA product. We investigated the roles of active site residues in CoA binding in acetyl-CoA synthetase (Acs) and a medium-chain acyl-CoA synthetase (Macs) that uses 2-methylbutyryl-CoA. Three highly conserved residues, Arg¹⁹³, Arg⁵²⁸, and Arg⁵⁸⁶ of <i>Methanothermobacter thermautotrophicus</i> Acs (Acs_Mt), are predicted to form important interactions with the 5'- and 3'-phosphate groups of CoA. Kinetic characterization of Acs_Mt variants altered at each of these positions indicates these Arg residues play a critical role in CoA binding and c2023-01-01T00:00:00Z2023 Jul2026-04-08T13:40:23.4Z2025-04-06T16:25:02.57ZS-EPMC104554773762950010.3390/life13081643