Abstract
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 μmol·kg-1 in brain to 1.4 μmol·kg-1 in kidney. We also show that N-acetylcysteine, a widely-used antioxidant, is a poor substrate for MPST and is 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 μm, whereas 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 MPST2 isoforms are kinetically indistinguishable and that thioredoxin modulates the MPST-catalyzed reaction in a physiologically-relevant concentration range.
Highlights
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
We conclude that the MPST1 and MPST2 isoforms are kinetically indistinguishable and that thioredoxin modulates the MPST-catalyzed reaction in a physiologically-relevant concentration range
Human MPST has two rhodanese-like domains, and Cys268/ Cys248 in the C-terminal domain of MPST1/MPST2 serves as the persulfide carrier [10]. 3-MP is derived via transamination of cysteine and ␣-ketoglutarate catalyzed by gluta
Summary
Mine oxoglutarate transaminase (GOT, Fig. 1B) involved in the malate-aspartate shuttle, which has cysteine oxoglutarate transaminase activity. In the first half-reaction, the sulfur atom is transferred from 3-MP to MPST, resulting in a cysteine persulfide intermediate in the active site. In the alternative SH-transfer pathway (Fig. 2B, bottom), the free energy changes for a concerted sulfur and proton transfer step were computed to be unfavorable with a single high-energy barrier In this second SH transfer mechanism, Ser250 was predicted to play a minor role. We have compared the kinetic properties of the MPST1 and MPST2 variants with cysteine, NAC, and thioredoxin and assessed the contributions of the catalytic triad residues to the reaction mechanism. We determined the concentration of 3-MP in murine liver, kidney, and brain and used the kinetic data to simulate the relative importance of physiologically-relevant acceptors to the MPST-dependent sulfur transfer reaction
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