Ferrous Human Cystathionine β-Synthase Loses Activity during Enzyme Assay Due to a Ligand Switch Process^†

Cystathionine β-synthase (CBS) is a pyridoxal-5‘-phosphate-dependent enzyme that catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS also contains a heme cofactor that has been proposed to allosterically regulate enzyme activity via the heme redox state, with Fe^II CBS displaying approximately half the activity of Fe^III CBS in vitro. The results of this study show that human Fe^II CBS spontaneously loses enzyme activity over the course of a 20 min enzyme assay. Both the full-length 63-kDa and truncated 45-kDa form of CBS slowly and irreversibly lose activity upon reduction to the Fe^II form. Additionally, electronic absorption spectroscopy reveals that Fe^II CBS undergoes a heme ligand exchange to Fe^II CBS424 when the enzyme is incubated at 37 °C and pH 8.6. The addition of enzyme substrates or imidazole has a moderate effect on the rate of the ligand switch, but does not prevent conversion to the inactive species. Time-dependent spectroscopic data describing the conversion of Fe^II CBS to Fe^II CBS424 were fitted to a three-state kinetic model. The resultant rate constants were used to fit assay data and to estimate the activity of Fe^II CBS prior to the ligand switch. Based on this fit it appears that Fe^II CBS initially has the same enzyme activity as Fe^III CBS, but Fe^II CBS loses activity as the ligand switch proceeds. The slow and irreversible loss of Fe^II CBS enzyme activity in vitro resembles protein denaturation, and suggests that a simple regulatory mechanism based on the heme redox state is unlikely.