Enzymes that control the rate-limiting steps in biosynthetic pathways are often subject to intricate regulation, ensuring their activity aligns with cellular energy demands. Post-translational modifications play a crucial role in this by altering an enzyme's conformational landscape and, consequently, its activity. Argininosuccinate synthetase (ASS1), a pivotal enzyme in both arginine biosynthesis and the urea cycle, has been previously reported to be inhibited by acetylation at lysine residues 165 and 176, a modification conferred by the CLOCK protein. While earlier studies showed that mutations mimicking lysine acetylation (K165Q and K176Q) resulted in lower ASS1 activity, the Michaelis-Menten constants were not determined, and critically, no structural information was available to explain the mechanism of this inhibition. In this study, we aim to address these gaps. We will first employ steady-state kinetic assays to precisely quantify the changes in activity for ASS1 K165Q/K176Q mutants. Furthermore, we will utilize cryo-electron microscopy (cryo-EM) to determine the high-resolution structure of these modified ASS1 variants. This structural analysis will provide crucial insights into the molecular mechanism by which acetylation diminishes ASS1 activity including a deeper inspection of the enclosing active site loop which harbors K165 and K176. Uncovering this mechanism is vital for unraveling how this specific PTM, and potentially others, modulate ASS1 function, offering broader implications for how loop conformations regulate metabolic enzymes.
Gurung et al. (Sun,) studied this question.