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J Biol Chem. 2018 Aug 10;293(32):12429-12439. doi: 10.1074/jbc.RA118.004096. Epub 2018 Jul 6.

Mechanism-based inhibition of human persulfide dioxygenase by γ-glutamyl-homocysteinyl-glycine.

Author information

1
From the Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109.
2
Inorganic Chemistry-Bioinorganic Chemistry, Ruhr University Bochum, 44801 Bochum, Germany, and.
3
the Department of Biochemistry and the Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588.
4
From the Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109, rbanerje@umich.edu.

Abstract

Hydrogen sulfide (H2S) is a signaling molecule with many beneficial effects. However, its cellular concentration is strictly regulated to avoid toxicity. Persulfide dioxygenase (PDO or ETHE1) is a mononuclear non-heme iron-containing protein in the sulfide oxidation pathway catalyzing the conversion of GSH persulfide (GSSH) to sulfite and GSH. PDO mutations result in the autosomal-recessive disorder ethylmalonic encephalopathy (EE). Here, we developed γ-glutamyl-homocysteinyl-glycine (GHcySH), in which the cysteinyl moiety in GSH is substituted with homocysteine, as a mechanism-based PDO inhibitor. Human PDO used GHcySH as an alternative substrate and converted it to GHcy-SO2H, mimicking GS-SO2H, the putative oxygenated intermediate formed with the natural substrate. Because GHcy-SO2H contains a C-S bond rather than an S-S bond in GS-SO2H, it failed to undergo the final hydrolysis step in the catalytic cycle, leading to PDO inhibition. We also characterized the biochemical penalties incurred by the L55P, T136A, C161Y, and R163W mutations reported in EE patients. The variants displayed lower iron content (1.4-11-fold) and lower thermal stability (1.2-1.7-fold) than WT PDO. They also exhibited varying degrees of catalytic impairment; the kcat/Km values for R163W, L55P, and C161Y PDOs were 18-, 42-, and 65-fold lower, respectively, and the T136A variant was most affected, with a 200-fold lower kcat/Km Like WT enzyme, these variants were inhibited by GHcySH. This study provides the first characterization of an intermediate in the PDO-catalyzed reaction and reports on deficits associated with EE-linked mutations that are distal from the active site.

KEYWORDS:

dioxygenase; enzyme kinetics; enzyme mechanism; ethylmalonic acid; ethylmalonic encephalopathy; hydrogen sulfide; iron; persulfide dioxygenase; sulfide oxidation

PMID:
29980601
PMCID:
PMC6093238
[Available on 2019-08-10]
DOI:
10.1074/jbc.RA118.004096

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