Characterization of the heme and pyridoxal phosphate cofactors of human cystathionine beta-synthase reveals nonequivalent active sites

Cystathionine beta-synthase is an unusual enzyme that requires the cofactor s heme and pyridoxal phosphate (PLP) to catalyze the condensation of homocy steine and serine to generate cystathionine. This transsulfuration reaction represents one of two major cellular routes for detoxification of homocyst eine, which is a risk factor for atherosclerosis. While the beta-replacemen t reaction catalyzed by this enzyme suggests a role for the pyridoxal phosp hate, the role of the heme is uncertain. In this study we have examined the effect of changing one of the ligands to the heme on the activity of the e nzyme. Binding of carbon monooxide results in the displacement of a thiolat e ligand to the ferrous heme, and is accompanied by complete loss of cystat hionine beta-synthase activity. Furthermore, inhibition by CO is competitiv e with respect to homocysteine, providing the first indication that the hom ocysteine binding site is in the proximity of heme. Binding of both CO and cyanide to ferrous cystathionine beta-synthase occurs in two distinct isoth erms and indicates that the hemes are nonequivalent. We have employed fluor escence spectroscopy to characterize the bound PLP and its interaction with serine. PLP bound to cystathionine beta-synthase is weakly fluorescent and exists as a mixture of the protonated and unprotonated tautomers. Reaction with hydroxylamine releases the oxime and greatly enhances the associated fluorescence. Binding of serine is accompanied by a shift to the unprotonat ed tautomer of the external aldimine as well as the appearance of a new flu orescent species at similar to 400 nm that could be due to the aminoacrylat e or to a gemdiamine intermediate. These data provide the first characteriz ation of the PLP bound to cystathionine beta-synthase. Treatment of cystath ionine beta-synthase with hydroxylamine releases two PLPs after 1 day and r esults in complete loss of activity. Incubation for an additional 3-4 days results in the release of two more PLPs. These data lead us to revise the P LP stoichiometry to 4 per tetramer, and to the conclusion that the heme and PLP sites in cystathionine beta-synthase are nonequivalent.