The instrumentation in this case also varied with the nucleophilic substrate. In both the case of CysO-COSH and sodium sulfide, the aminoacrylate intermediate was pre-formed by treatment of CysM (14 μM) with a solution of O-phospho-L-serine (11 μM) under single turnover conditions. This was then mixed with solutions of CysO-COSH (25 – 250 μM) or sodium sulfide (100 μM — 15 mM). In the case of CysO-COSH, the lowest concentration used (25 μM) was not under pseudo-first order conditions. For CysO-COSH, the aminoacrylate and CysO-COSH solutions were mixed rapidly in a stopped-flow device and the decay of absorbance at 465 nm due to reaction of the aminoacrylate intermediate was observed. In the case of sodium sulfide, the aminoacrylate and bisulfide-containing solutions were mixed manually and the decay of absorbance was recorded in the presence of the reductant TCEP (2 mM). In both cases these decays were fit by non-linear regression to single exponential functions to yield the first order rate constants for quenching of the aminoacrylate under the varying conditions of nucleophile concentration. These rate constants were then plotted as a function of nucleophile concentration. In the case of CysO-COSH the data thus obtained were fit to a line, whereas in the case of bisulfide the dependence of the first order rate constants on the nucleophile concentration was fit to a hyperbola.