Modulation of Homocysteine Toxicity by S-Nitrosothiol Formation: A Mechanistic Approach

Abstract

The metabolic conversion of homocysteine (HCYSH) to homocysteine thiolactone (HTL) has been reported as the major cause of HCYSH pathogenesis. It was hypothesized that inhibition of the thiol group of HCYSH by S-nitrosation will prevent its metabolic conversion to HTL. The kinetics, reaction dynamics, and mechanism of reaction of HCYSH and nitrous acid to produce S-nitrosohomocysteine (HCYSNO) was studied in mildly to highly acidic pHs. Transnitrosation of this non-protein-forming amino acid by S-nitrosoglutathione (GSNO) was also studied at physiological pH 7.4 in phosphate buffer. In both cases, HCYSNO formed quantitatively. Copper ions were found to play dual roles, catalyzing the rate of formation of HCYSNO as well as its rate of decomposition. In the presence of a transition-metal ions chelator, HCYSNO was very stable with a half-life of 198 h at pH 7.4. Nitrosation by nitrous acid occurred via the formation of more powerful nitrosating agents, nitrosonium cation (NO(+)) and dinitrogen trioxide (N(2)O(3)). In highly acidic environments, NO(+) was found to be the most effective nitrosating agent with a first-order dependence on nitrous acid. N(2)O(3) was the most relevant nitrosating agent in a mildly acidic environment with a second-order dependence on nitrous acid. The bimolecular rate constants for the direct reactions of HCYSH and nitrous acid, N(2)O(3), and NO(+) were 9.0 x 10(-2), 9.50 x 10(3), and 6.57 x 10(10) M(-1) s(-1), respectively. These rate constant values agreed with the electrophilic order of these nitrosating agents: HNO(2) < N(2)O(3) < NO(+). Transnitrosation of HCYSH by GSNO produced HCYSNO and other products including glutathione (reduced and oxidized) and homocysteine-glutathione mixed disulfide. A computer modeling involving eight reactions gave a good fit to the observed formation kinetics of HCYSNO. This study has shown that it is possible to modulate homocysteine toxicity by preventing its conversion to a more toxic HTL by S-nitrosation.

State of the art paper Relationship between paraoxonase and homocysteine: crossroads of oxidative diseases

“…The condition of excess Hcy can be exacerbated by inadequate intake of folic acid, vitamin B 12 , and vitamin B 6 as well as by the allelic variation in genes encoding enzymes participating in Hcy metabolism [31, 32]. …”

Kinetics of S-Nitrosation Processes in Aqueous Polymer Solution for Controlled Nitric Oxide Loading: Toward Tunable Biomaterials

“…33 The 545 nm peak was monitored to measure the formation of the GSNO product in these studies instead of the 335 nm peak, despite a lower extinction coefficient, because of a lack of interfering species (NO 2 − , HNO 2 ) at 545 nm. 34 The stability of GSNO is important as the nitrosothiol moiety decomposition will not significantly compete with the rate of its formation. We investigated two commonly reported nitrosating agents when used in a fixed molar excess relative to glutathione: nitrous acid (HNO 2 ) and t-butyl nitrite.…”

“…For the HNO 2 nitrosation of thiols described herein, a first-order dependence on the thiol has been reported. 34,35 We experimentally monitor the formation of S-nitrosoglutathione, however, we report the kinetics in terms of the disappearance of the thiol reactant. Theoretically, the disappearance of the thiol could be attributed to either the formation of the S-nitrosated product or possible disulfide formation.…”

Biomarkers of nitro-oxidation and oxidative stress

“…It is therefore essentially a physiological event which may exert protective actions. Indeed, Snitrosation of homocysteine has been reported to prevent its conversion to its toxic thiolactone metabolite [20]. Deleterious effects of S-nitrosation only seem to appear when the R-SNO groups cannot be reduced to R-SH.…”