TY - JOUR
T1 - Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically-relevant concentrations
AU - Yadav, Pramod Kumar
AU - Vitvitsky, Victor
AU - Carballal, Sebastián
AU - Seravalli, Javier
AU - Banerjee, Ruma
N1 - Funding Information:
This work was supported in part by National Institutes of Health Grant HL58984 and American Heart Association Grant 14POST18760003 (to P. K. Y.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Fig. S1. 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed. Tel.: 734-615-5238; E-mail: rbanerje@umich.edu. 3The abbreviations used are: H2S, hydrogen sulfide; MPST, mercaptopyru-vate sulfur transferase; 3-MP, 3-mercaptopyruvate; GSH, glutathione; NAC,
Publisher Copyright:
© 2020 Yadav et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2020/5/8
Y1 - 2020/5/8
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.RA120.012616
DO - 10.1074/jbc.RA120.012616
M3 - Article
C2 - 32179647
AN - SCOPUS:85084721251
SN - 0021-9258
VL - 295
SP - 6299
EP - 6310
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 19
ER -