Human Citrate Synthase Post‐Translational Modification Mimics and Molecular Dynamic Simulations Demonstrate Attenuation of Acetyl‐ <scp>CoA</scp> / <scp>CoA</scp> Binding

Human citrate synthase (hCS) is a mitochondrial enzyme that catalyzes the aldol condensation of acetyl coenzyme A (AcCoA) to oxaloacetate to form citrate in the TCA cycle. CS activity is important for aerobic exercise performance and basic metabolic function as a housekeeping enzyme. It has been shown through several mass spectrometry-based physiological studies that CS is post-translationally modified (PTM) on numerous residues via acetylation, phosphorylation, and methylation reactions. Few follow-up studies have been reported on the impact of PTMs on CS activity. Thus, we kinetically characterized several hCS PTM mimics near and distant from the active site by site-directed mutagenesis coupled with steady-state kinetics. Most modifications had a negative impact on AcCoA k_cat/K_m but to a much lesser extent on oxaloacetate k_cat/K_m. Most notably, the K393 acetylation mimic, K393Q displays an increase in K_m for AcCoA relative to WT by about 30-fold, with no significant change in k_cat. To complement our kinetic analyses, we performed molecular dynamics simulations on 26 PTM and mutant CS-substrate complexes, providing a combined kinetic and MD simulation approach. Among the MD results, CS K393AcK showed the greatest reduction in AcCoA/CoA binding.