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Open AccessArticle

Evidence for an Allosteric S-Nitrosoglutathione Binding Site in S-Nitrosoglutathione Reductase (GSNOR)

1
Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Ave., Windsor, ON N9B 3P4, Canada
2
Apotex Inc., 150 Signet Dr., North York, ON N9B 3P4, Canada
3
Department of Chemistry, York University, 4700 Keele Street, Toronto, ON N9B 3P4, Canada
*
Author to whom correspondence should be addressed.
Antioxidants 2019, 8(11), 545; https://doi.org/10.3390/antiox8110545
Received: 18 September 2019 / Revised: 3 November 2019 / Accepted: 12 November 2019 / Published: 13 November 2019
(This article belongs to the Special Issue NO(NOx) and H2S)
Current research has identified S-nitrosoglutathione reductase (GSNOR) as the central enzyme for regulating protein S-nitrosylation. In addition, the dysregulation of GSNOR expression is implicated in several organ system pathologies including respiratory, cardiovascular, hematologic, and neurologic, making GSNOR a primary target for pharmacological intervention. This study demonstrates the kinetic activation of GSNOR by its substrate S-nitrosoglutathione (GSNO). GSNOR kinetic analysis data resulted in nonhyperbolic behavior that was successfully accommodated by the Hill–Langmuir equation with a Hill coefficient of +1.75, indicating that the substrate, GSNO, was acting as a positive allosteric affector. Docking and molecular dynamics simulations were used to predict the location of the GSNO allosteric domain comprising the residues Asn185, Lys188, Gly321, and Lys323 in the vicinity of the structural Zn2+-binding site. GSNO binding to Lys188, Gly321, and Lys323 was further supported by hydrogen–deuterium exchange mass spectroscopy (HDXMS), as deuterium exchange significantly decreased at these residues in the presence of GSNO. The site-directed mutagenesis of Lys188Ala and Lys323Ala resulted in the loss of allosteric behavior. Ultimately, this work unambiguously demonstrates that GSNO at large concentrations activates GSNOR by binding to an allosteric site comprised of the residues Asn185, Lys188, Gly321, and Lys323. The identification of an allosteric GSNO-binding domain on GSNOR is significant, as it provides a platform for pharmacological intervention to modulate the activity of this essential enzyme. View Full-Text
Keywords: S-nitrosoglutathione reductase; GSNOR; hydrogen–deuterium exchange mass spectroscopy; allosteric site for S-nitrosoglutathione; GSNO; docking and molecular dynamics simulations S-nitrosoglutathione reductase; GSNOR; hydrogen–deuterium exchange mass spectroscopy; allosteric site for S-nitrosoglutathione; GSNO; docking and molecular dynamics simulations
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MDPI and ACS Style

Fontana, K.; Onukwue, N.; Sun, B.-L.; Lento, C.; Ventimiglia, L.; Nikoo, S.; Gauld, J.W.; Wilson, D.J.; Mutus, B. Evidence for an Allosteric S-Nitrosoglutathione Binding Site in S-Nitrosoglutathione Reductase (GSNOR). Antioxidants 2019, 8, 545.

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