Ferrous human cystathionine beta-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 FeII CBS displaying approximately half the activity of FeIII CBS in vitro. The results of this study show that human FeII 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 FeII form. Additionally, electronic absorption spectroscopy reveals that FeII CBS undergoes a heme ligand exchange to FeII 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 FeII CBS to FeII 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 FeII CBS prior to the ligand switch. Based on this fit it appears that FeII CBS initially has the same enzyme activity as FeIII CBS, but FeII CBS loses activity as the ligand switch proceeds. The slow and irreversible loss of FeII CBS enzyme activity in vitro resembles protein denaturation, and suggests that a simple regulatory mechanism based on the heme redox state is unlikely.
Cherney
Melisa M
MM
Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Pazicni
Samuel
S
Frank
Nina
N
Marvin
Katherine A
KA
Kraus
Jan P
JP
Burstyn
Judith N
JN
D000494:Allosteric Regulation; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D005296:Ferrous Compounds; D007093:Imidazoles; D008024:Ligands; D006418:Heme; C029899:imidazole; D003541:Cystathionine beta-Synthase