H2S Production

This post was inspired by a review on fatty liver disease that listed four enzymes responsible for H₂S production.

Transsulfuration Pathway to H₂S in NAFLD [1]

The authors are interested in non alcoholic fatty liver disease because 25% of the world’s population of humans is estimated to have some form of this disease.

The following is a rather extensive table from the fatty live publication. [1]. Enzymes that will be expanded upon are in bold red. Some of the enzymes in the above cartoon were not in the table. These enzymes were added and referenced accordingly.

Enzyme Name	Gene Name	Cofactor	Tissue Expression
Adenosylhomocysteinase (SAHH)	AHCY	NAD+	Low tissue specificity. High in liver, pancreas and kidney, endocrine tissue, female and male tissue.
Betaine-homocysteine S methyltransferase (BHMT)—Two genes	BHMT BHMT2	Zinc	High tissue specificity. High in liver, kidney, and urinary tract.
Cystathionine beta-synthase (CBS)	CBS	Pyridoxal 5′-phosphat (B6)	High tissue specificity. High in liver and pancreas. Some in heart and brain.
Cystathionine gamma-lyase (CSE)	CTH	Pyridoxal 5′-phosphat (B6)	High tissue specificity. High in liver, female tissue, endocrine tissue. Some in the pancreas, brain, kidneys.
cysteine transaminase			in bovine brain a interesting study that got lost in 1989
3-mercaptopyruvate sulfurtransferase (MPST)	MPST	none [6]	kidney, lung, heart, brain, glia [6]
Cysteine dioxygenase (CDO)	CDO1	Iron	High tissue specificity. Highly expressed in liver and placenta. Some expression in heart, adipose tissue, brain, and pancreas.
Cysteine sulfinic acid decarboxylase (CSD)	CSAD	Pyridoxal 5′-phosphat (B6)	Low tissue specificity. Expressed in liver, gastrointestinal (GI) tract, brain female and male tissue, muscle tissue, and adipose tissue.
Glutathione peroxidase (GPO)—several genes	GPX 1–8		low tissue specificity. high in the liver, gallbladder and GI tract.
Glutathione reductase (GR)	GSR	FAD	Low tissue specificity. Highly expressed in liver, pancreas, GI tract, endocrine tissue, kidney, female and male tissue.
Glutathione synthetase (GS)	GSS	Magnesium	Low tissue specificity. Highly expressed in brain, endocrine tissue, GI tract, kidney and liver.
Glutamate-cysteine ligase (GCL)	GCLM GCLC		Methionine synthase Highly expressed in the liver.
Glycine N-methyltransferase (GNMT)	GNMT		High tissue specificity. Highly expressed in liver and pancreas. Some expression in brain, GI tract, and kidney.
Methionine adenosyltransferase (MAT)	MAT1A	Potasium Magnesium	Highly expressed in liver, pancreas. Some expression lungs and female and male tissue.
Methionine synthase (MS)	MTR	Cobalamin (B12) Zinc	high in pancreas, heart, brain, skeletal muscle and placenta. lower levels in lung, liver, and kidney.
Methylenetetrahydrofolate reductase (MTHFR)	MTHFR	FAD	Low tissue specificity. Highly expressed in female and male tissue, GI tract and kidney. Some expression in liver.
Serine hydroxymethyltransferase	SHMT1 SHMT2	Pyridoxal 5′-phosphat (B6)	High tissue specificity. High liver and kidney. Some lungs, brain, pancreas, and GI tract.

Cystathionine gamma-lyase (CSE)

Some of this information is from UniProt. CSE catalyzes the last step in the trans-sulfuration pathway from L-methionine to L-cysteine in a pyridoxal-5′-phosphate (PLP)-dependent manner. PLP is the active form of vitamin B6. The L-cystathionine molecule is cleaved into L-cysteine, ammonia and 2-oxobutanoate. Some important side reactions include:

• H₂O + L-cysteine = H⁺ + H₂S + NH₄⁺ + pyruvate
• H₂O + L-homocysteine = 2-oxobutanoate + H⁺ + H₂S + NH₄⁺
• L-homoserine = 2-oxobutanoate + NH₄⁺

The H₂S, hydrogen sulfide, may play an important regulatory role via sulfination whereby Acts as a cysteine-protein sulfhydrase by mediating sulfhydration, which consists of converting -SH groups into -SSH on specific cysteine residues of target proteins. UniProt annotation lists these as: GAPDH, PTPN1 and NF-kappa-B subunit RELA. The Werge review had more to say about transcriptional control of CSE. These transcription factors include: FXR, GPBAR-1, SP1, and NRF2. [1]

Cystathionine beta-synthase (CBS)

The CBS gene that codes for cystathionine β-synthase is located on the long arm of hman chromosome 21, the same chromosome that exists in triplicate in Down’s Syndrome.

CBS in Down’s Syndrome [2]

It is very difficult to get to the bottom of which of many reactions CBS catalyzes and if CBE is an obligate partner. The CBE gene is located on chromosome 1 so it is not co duplicated in Down’s Syndrome.

Panagaki and coaluthors isolated fibroblasts from volunteers with Down’s Syndrome for the purpose assessing mitochondria function. DS fibroblasts showed higher CBS expression than control fibroblasts. CBS was shown to be localized in the cytosol and mitochondria. DS fibroblasts produced more H₂S and polysulfide and exhibited a profound suppression of mitochondrial electron transport, oxygen consumption, and ATP generation. [2] When CBS over activity was corrected by reducing CBS expression with siRNA and pharmacological inhibition, mitochondrial function was restores. [2]

CBS regulation by glutathionylation [3]

Glutathionylation is the covalent attachment of the thiol group glutathione to a protein cysteine via an S=S double bond. GSH, being rather bulky, can change the structure of an enzyme making it more or less active.

EC 2.6.1.3: cysteine transaminase, cysteine aminotransferase (CAT)

EC 2.6.1.3 links to a cite telling us that cysteine transaminase is the official name of the enzyme that catalyzes this reaction:

2-oxoglutarate + L-cysteine <=> 2-oxo-3-sulfanylpropanoate + L-glutamate

Cysteine amino transferase is an accepted name. Note: 2-oxo-3-sulfanylpropanoate is the official name for 3-mercaptopyruvate.

found in bovine brain, [4]

This enzyme was purified from bovine brain in 1989. The activity was found in cerebral white and gray matter. The cerebellum had about half the activity of the cerebrum. No transaminase activity was found in the pons-medulla oblongata. [4] The standard assay solution contained 10#mol of Saminoethylcysteine, 0.5/amol of ~-keto-7-methiolbutyric acid and variable amounts of enzyme. [4] As a side note, an active site thiol appears to be involved in catalysis.

CAT does not act alone [5]

A group from Japan looked at H₂S production in rat liver, brain, spinal cord dorsal root ganglion, and neuron like PC12 cells with and without factors to induce differentiation into a neuron like phenotype. These authors proceeded with the premise that CAT/MPST are an inseparable pair just as are CSE/CBS.

• CSE protein was only detected in the liver. Some CSE like H₂S production DRG and brain. Rat liver elicited production using a CSE substrate. H₂S production was seen in the liver.
• MItochondria and cytosolic CAT were observed in PC12 cells, DRG, and the brain. The large variant of MPST was observed in all four. The small version of MPST was not observed in PC12 cells. In the presence of a-KG, CAT converts L-cysteine to
  3MP. MPST metabolizes to H2Sin the presence of sulfur acceptors, such as DTT With a-KG and DTT included in the reaction buffer at pH 8.5, considerable amounts of H2S were produced from L-cysteine not only in the brain but also in undifferentiated PC12 cells,
• In undifferentiated PC12 cells ramping a-ketoglutarate (a-KG) (0.01–1 mM) and dithiothreitol (0.1–10 mM) increased H₂S production. dihydrolipoic acid (0.1–3 mM) hand a modest effect. A CSE inhibitor had no effect on H₂S production whereas CAT and MPST inhibitors did inhibit.
• In PC12 cells MPST silencing RNA decreased H₂S production.
• MPST localized to the mitochondria. CAT
  

In summary, cysteine amino transferase / cysteine transaminase plays a role in H2S production by generating 3-mercaptoproponate / 2-oxo-3-sulfanylpropanoate to hand off it its partner MPST.

3-mercaptopyruvate sulfurtransferase (MPST)

Noriyuki Nagahara of Nippon Medical School in Tokyo Japan has written an excellent review on the rat isoform of MPST. MPST basically has a catalytic and surface reduced thiols that can catalyze oxidation of protein and small molecule thiols. [6] Dr. Nagahara has been working on MPST for over a quarter of a century. This post will not get into the discussion of sulfate and sulfite.

Figure 1 ” Summary of four functions of MPST. Two definite and two possible functions are shown. In the antioxidative function, the catalytic site Cys²⁴⁷ (MPST–S), which acts as a redox‐sensing switch, is oxidized to MPST–SO (cysteine sulfenate) and then reduced by reduced Trx. Moreover, a disulfide bond formed between MPST–S (red) and MPST–S (blue) (MPST–Cys¹⁵⁴ and MPST–Cys²⁶³, respectively) acts as another redox‐sensing switch. In hydrogen sulfide and polysulfide (H₂S_n) production, a sulfur of the donor substrate is first transferred to MPST–S⁻ to form MPST–S_n–S⁻ (persulfurated MPST, reaction intermediate). One pathway proceeds via the reduction of persulfide or polysulfide formed at the sulfur‐acceptor substrate (RS′S_n) by reduced Trx. The other pathway proceeds via the reduction of reaction intermediate (MPST–S_n–S⁻) by reduced Trx. In sulfur oxide production, MPST–S–S⁻ is oxidized to MPST–S–SO⁻ (thiosulfenyl MPST) and reduced by reduced Trx to release SO.”

Dr. Nagahara did devoted a small amount of his review to the production of H₂S.

Another MPST review comes out of College of Pharmacy, University of Minnesota. These authors are less into catalytic mechanisms than the global view. They gave a nice review of cysteine amino trasferase as being the source of 3-mercaptopyruvate from L-cysteine and daimine oxidase producing mercaptopyruvate from D-cysteine, [7] The authors reiterated the role of thioredoxin as being the actual producer of H₂S.

Exhausted and brief summary

The four enzymes in pairs CBS/CSE and CAT/MPST act together to generate H₂S.

References

1. Werge MP, McCann A, Galsgaard ED, Holst D, Bugge A, Albrechtsen NJW, Gluud LL. The Role of the Transsulfuration Pathway in Non-Alcoholic Fatty Liver Disease. J Clin Med. 2021 Mar 5;10(5):1081. PMC free article
2. Panagaki T, Randi EB, Augsburger F, Szabo C. Overproduction of H₂S, generated by CBS, inhibits mitochondrial Complex IV and suppresses oxidative phosphorylation in Down syndrome. Proc Natl Acad Sci U S A. 2019 Sep 17;116(38):18769-18771. free article
3. Niu WN, Yadav PK, Adamec J, Banerjee R. S-glutathionylation enhances human cystathionine β-synthase activity under oxidative stress conditions. Antioxid Redox Signal. 2015 Feb 10;22(5):350-61. PMC free article
4. Pensa B, Achilli M, Fontana M, Caccuri AM, Cavallini D. S- aminoethyl- l -cysteine transaminase from bovine brain: purification to homogeneity and assay of activity in different regions of the brain. Neurochem Int. 1989;15(3):285-91. Sci-Hub free article
5. Miyamoto R, Otsuguro K, Yamaguchi S, Ito S. Contribution of cysteine aminotransferase and mercaptopyruvate sulfurtransferase to hydrogen sulfide production in peripheral neurons. J Neurochem. 2014 Jul;130(1):29-40. free article
6. Nagahara N. Multiple role of 3-mercaptopyruvate sulfurtransferase: antioxidative function, H₂ S and polysulfide production and possible SO_x production. Br J Pharmacol. 2018 Feb;175(4):577-589. PMS free article
7. Rao SP, Dobariya P, Bellamkonda H, More SS. Role of 3-Mercaptopyruvate Sulfurtransferase (3-MST) in Physiology and Disease. Antioxidants (Basel). 2023 Mar 1;12(3):603. PMC free article