Production of the neuromodulator H₂S by cystathionine β-synthase via the condensation of cysteine and homocysteine

Hydrogen sulfide (H₂S) has been observed in relatively high concentrations in the mammalian brain and has been shown to act as a neuromodulator. However, there is confusion in the literature regarding the actual source of H₂S production. Reactions catalyzed by the cystathionine β-synthase enzyme (CBS) are one possible source for the production of H₂S. Here we show that the CBS enzyme can efficiently produce H₂S via a β-replacement reaction in which cysteine is condensed with homocysteine to form cystathionine and H₂S. The production of H₂S by this reaction is at least 50 times more efficient than that produced by hydrolysis of cysteine alone via β-elimination. Kinetic studies demonstrate that the K_m and K_cat for cysteine is 3-fold higher and 2-fold lower, respectively, than that for serine. Consistent with these data, in vitro reconstitution studies show that at physiologically relevant concentrations of serine, homocysteine, and cysteine, about 5% of the cystathionine formed is from cysteine. We also show that AdoMet stimulates this H₂S producing reaction but that there is no evidence for stimulation by calcium and calmodulin as reported previously. In summary, these results confirm the ability of CBS to produce H₂S, but show in contrast to prior reports that the major mechanism is via β-replacement and not cysteine hydrolysis. In addition, these studies provide a biochemical explanation for the previously inexplicable homocysteine-lowering effects of N-acetylcysteine treatments in humans.