Structure-function relationship in S-nitrosoglutathione reductase and the development of fluorogenic pseudo-substrates

S-nitrosation is the attachment of a nitric oxide moiety to the thiol side chain of cysteine. S-nitrosoglutathione (GSNO) acts as a bioactive reservoir for NO to maintain an equilibrium in the concentration of NO in the body. Due to this, the study of the enzyme S-nitrosoglutathione reductase has of great interest because of its ability to metabolize GSNO. S-nitrosoglutathione reductase’s activity has been linked to a number of human diseases. Chapter 1 of this thesis presents a proposed allosteric binding domain on GSNOR. Positive cooperativity (sigmoidal deviation) was observed from steady state analysis of GSNOR which indicated an affinity for the binding of GSNO at this site. The presence of such a site was further supported by Molecular docking simulations and HDX-MS which showed that the amino acids Gly321, Lys323, Asn185 and Lys188 interact with molecules bound at this site. Chapter two introduces four reagents that can function as probes or pseudo-substrates for the monitoring of enzymatic activity as well as measuring concentrations of free thiols in vitro and live cells. These reagents are N,N-di(thioamido-fluoresceinyl)-cystine (DTFCys2), N,N-di(thioamido-fluoresceinyl)-homocystine (DTFHCys2), N-amido-O-aminobenzoyl-S-nitrosoglutathione (AOASNOG), and N-thioamido-fluoresceinyl-S-nitroso-glutathione (TFSNOG). They are easy to prepare and purity and can be used in various applications

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

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