Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically relevant concentrations.

3-Mercaptopyruvate sulfur transferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate (3-MP) and transfers sulfane sulfur from an enzyme-bound persulfide intermediate to thiophilic acceptors such as thioredoxin and cysteine. Hydrogen sulfide (H2S), a signaling molecule implicated in many physiological processes, can be released from the persulfide product of the MPST reaction. Two splice variants of MPST, differing by 20 amino acids at the N-terminus, give rise to the cytosolic MPST1 and mitochondrial MPST2 isoforms. Here, we characterized the poorly studied MPST1 variant and demonstrated that substitutions in its Ser-His-Asp triad, proposed to serve a general acid-base role, minimally affect catalytic activity. We estimated the 3-MP concentration in murine liver, kidney and brain tissues, finding that it ranges from 0.4 mumol*kg-1 in brain to 1.4 mumol*kg-1 in kidney. We also show that N-acetylcysteine, a widely used antioxidant, is a poor substrate for MPST and unlikely to function as a thiophilic acceptor. Thioredoxin exhibits substrate inhibition, increasing the KM for 3-MP ~15-fold compared with other sulfur acceptors. Kinetic simulations at physiologically relevant substrate concentrations predicted that the proportion of sulfur transfer to thioredoxin increases ~3.5-fold as its concentration decreases from 10 to 1 microM, while the total MPST reaction rate increases ~7-fold. The simulations also predicted that cysteine is a quantitatively significant sulfane sulfur acceptor, revealing MPST's potential to generate low molecular weight persulfides. We conclude that the MPST1 and 2 isoforms are kinetically indistinguishable and that thioredoxin modulates the MPST-catalyzed reaction in a physiologically relevant concentration range.