Enzymes catalyzing rate-limiting steps in biosynthetic pathways often undergo complex allosteric regulation to match their activity to cellular energy demands. Protein interactions with small molecules can alter the underlying conformational landscape of enzymes to modulate their activity. Argininosuccinate synthetase (ASS1), a key enzyme in both arginine biosynthesis and the urea cycle, represents a potential target for allosteric modulation. Notably, the pesticide spinosyn A has been shown to increase ASS1 activity through a covalent modification at a site distant from the active site. However, the precise mechanism of this allosteric activation is unknown. In this study, steady-state kinetic assays and cryo-electron microscopy (cryo-EM) are utilized to define the conformational landscape of ASS1 under catalytic conditions and, further, to investigate the mechanism by which spinosyn A enhances ASS1 activity. Understanding this allosteric mechanism is crucial, particularly for cancer therapeutics, as ASS1 inhibition is considered a promising adjunct to arginine depletion therapies using arginine deiminase.
Michelle J. Von Merta-Sustarich (Sun,) studied this question.