〈Introduction〉Cystathionine γ-lyase (CSE) is an enzyme responsible for the biosynthesis of cysteine from cystathionine. It also has the β-lyase activity toward cystine to generate cysteine hydropersulfide (Cys-SSH). Notably, the chemical reactivity of Cys-SSH is thought to be involved in the catalytic activity of proteins via protein polysulfidation. In the present study, we investigated Cys-SSH could regulate CSE enzyme activity through its polysulfidation.
〈Method〉CSE enzyme activity (levels of enzymatically synthesized CysSSH) was determined with a fluorescent probe sulfane sulfur probe 4 (SSP4). Polysulfidated CSE was detected using modified biotin switch assay.
〈Results and discussion〉In vitro incubation of CSE with Na₂S₄ induced the inhibition of the enzyme, accompanied by its polysulfidation. Treatment with dithiothreitol reversed the polysulfidation and the subsequent inhibition. Similarly, in vitro incubation of CSE with CSE-enzymatically synthesized Cys-SSH resulted in the inhibition of the enzyme. We generated Na₂S₄-insensitive CSE mutants in that its specific Cys resides were mutated with valine. Furthermore, the mutant displayed a reduction in CSE polysulfidation by Na₂S₄ relative to the wild-type enzyme. Interestingly, the enzyme activities of Na₂S₄-insensitive CSE mutant were higher than that of wild-type. Thus, CysSSH is endogenously generated and auto-inhibits CSE enzyme activity via polysulfidation of its specific cysteine residues.