Ferrous human cystathionine β-synthase loses activity during enzyme assay due to a ligand switch process

Cystathionine beta-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 degrees 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.