Date of Award
Chemistry and Biochemistry
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Hydrogen sulfide (H2 S) produced in the reverse transsulfuration pathway is a significant signaling molecule. The two known pyridoxal-5'-phosphate (PLP)-dependent enzymes that catalyze H2 S generation in mammals are cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). The interaction between H2 S/CSE and NO/NOS systems, which have regulatory roles, has been recently discovered in mice. To study the effects of signaling molecules NOx on human recombinant cystathionine γ-lyase, recombinant CSE was incubated in the presence of low or high oxygen contents with NO donors diethylamine NONOate (DEANO) and S-Nitrosoglutathione (GSNO). The enzyme was then tested for H2 S production upon incubation with the CSE substrate, L-cysteine. A novel, real-time H2 S detection method using H2 S-permeable polydimethylsiloxane (PDMS) and thiol-reactive probe, Ellman's reagent, was utilized for enzyme activity measurement. These experiments reveal that the catalytic efficiency, Kcat /KM , of substrate was found to be significantly affected by NO incubation. NO incubation decreased the enzyme activity by lowering the binding affinity of the substrate to the enzyme. At 2 ppm or 16 ppm oxygen concentration, NO released from NO donor was proposed to directly modify protein residues such as cysteine, tryptophan and tyrosine. In addition, inhibition of NO production in RAW 264.7 macrophage can induce H 2 S production. These studies indicate a potential regulatory role of NO in H2 S production by either directly reacting with H2 S or modulating H2 S-producing pathways.
Wang, Jing Yuan Rebecca, "The Crosstalk between Hydrogen Sulfide and Nitric Oxide Signaling Pathways " (2012). Electronic Theses and Dissertations. 4849.