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

TL;DR

This study explores how post-translational modifications affect human citrate synthase activity and substrate binding using experiments and simulations.

Contribution

A combined kinetic and molecular dynamics approach to assess PTM effects on hCS function.

Findings

PTM mimics near the active site significantly reduced AcCoA binding efficiency.

K393 acetylation mimic (K393Q) increased AcCoA Km by 30-fold compared to wild-type.

Molecular dynamics simulations revealed reduced AcCoA/CoA binding in K393AcK.

Abstract

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 kcat/Km but to a much lesser extent on oxaloacetate kcat/Km. Most notably, the K393 acetylation mimic, K393Q displays an increase in Km for AcCoA relative to WT by about 30‐fold, with no significant change in kcat. 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.

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