Reactive Sulfur Species: Kinetics and Mechanism of the Hydrolysis of Cysteine Thiosulfinate Ester

The kinetics and mechanisms of the hydrolysis of cysteine thiosulfinate ester (CyS(O)SCy x−, x = 0–2) have been investigated by stopped-flow spectrophotometry between pH 6 and pH 14. The rate-limiting reaction of hydroxide is observed for pH < 13. More complicated kinetics are observed above pH 13, where the hydrolysis of CyS(O)SCy2− can be fast relative to subsequent reactions. The eventual products of hydrolysis are a 1:1 molar ratio of cystine (CySSCy) and cysteine sufinic acid (CySO2H) under all reaction conditions. The rate of hydrolysis is dependent upon the proton state of CyS(O)SCy x−. Furthermore, cysteine thiosulfonate ester (CyS(O)2SCy) was observed as an intermediate during the hydrolysis of CyS(O)SCy x− at lower pH. CyS(O)2SCy eventually hydrolyzes to give stoichiometric amounts of CySSCy and CySO2H. However, CySO2H is observed under some conditions for which hydrolysis of CyS(O)2SCy is relatively slow, thus suggesting multiple hydrolysis pathways for CyS(O)SCy x−. The mechanism up to the rate-limiting step is proposed to be as follows: CyS(O)SCy0 = H+ + CyS(O)SCy−, pK a3 = 7.32; CyS(O)SCy− = H+ + CyS(O)SCy2−, pK a4 = 7.92; CyS(O)SCy0 + OH− → products, P 0 k 0 = (5.0 ± 0.01) × 103 M−1 s−1; CyS(O)SCy− + OH− → products, P 1 k 1 = 60 ± 18 M−1 s−1; and CyS(O)SCy2− + OH− → products, P 2 k 2 = 0.36 ± 0.01 M−1 s−1, where P x is a constant (1 ≤ P x ≤ 3) that accounts for the partitioning between the possible hydrolysis pathways and the stoichiometries of their net reactions.