Journal of Biological Chemistry
Volume 293, Issue 7, 16 February 2018, Pages 2523-2533
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Enzymology
Allosteric control of human cystathionine β-synthase activity by a redox active disulfide bondAllosteric control of CBS activity

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Cystathionine β-synthase (CBS) is the central enzyme in the trans-sulfuration pathway that converts homocysteine to cysteine. It is also one of the three major enzymes involved in the biogenesis of H2S. CBS is a complex protein with a modular three-domain architecture, the central domain of which contains a 272CXXC275 motif whose function has yet to be determined. In the present study, we demonstrated that the CXXC motif exists in oxidized and reduced states in the recombinant enzyme by mass spectroscopic analysis and a thiol labeling assay. The activity of reduced CBS is ˜2–3-fold greater than that of the oxidized enzyme, and substitution of either cysteine in CXXC motif leads to a loss of redox sensitivity. The Cys272–Cys275 disulfide bond in CBS has a midpoint potential of −314 mV at pH 7.4. Additionally, the CXXC motif also exists in oxidized and reduced states in HEK293 cells under oxidative and reductive conditions, and stressing these cells with DTT results in more reduced enzyme and a concomitant increase in H2S production in live HEK293 cells as determined using a H2S fluorescent probe. By contrast, incubation of cells with aminooxyacetic acid, an inhibitor of CBS and cystathionine γ-lyase, eliminated the increase of H2S production after the cells were exposed to DTT. These findings indicate that CBS is post-translationally regulated by a redox-active disulfide bond in the CXXC motif. The results also demonstrate that CBS-derived H2S production is increased in cells under reductive stress conditions.

oxidation-reduction (redox)
redox regulation
hydrogen sulfide
disulfide
allosteric regulation
homocysteine
cystathionine beta synthase
sulfur metabolism

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This work was supported by Grant 31471086 from the National Natural Science Foundation of China, Grant 2017GY-143 from the Key Science and Technology Program of Shaanxi Province, China, Grant G2017KY0001 from the Fundamental Research Funds for the Central Universities of China, and Grant Z2017233 from the Seed Foundation of Innovation and Creation for Graduate Students in Northwestern Polytechnical University. The authors declare that they have no conflicts of interest with the contents of this article.

This article contains supporting text and Figs. S1–S3.

1

Both authors contributed equally to this work.