Stopped-flow kinetic analysis of the reaction catalyzed by the full-length yeast cystathionine β-synthase

Abstract

Cystathionine β-synthase found in yeast catalyzes a pyridoxal phosphate-dependent condensation of homocysteine and serine to form cystathionine. Unlike the homologous mammalian enzymes, yeast cystathionine β-synthase lacks a second cofactor, heme, which facilitates detailed kinetic studies of the enzyme because the different pyridoxal phosphate-bound intermediates can be followed by their characteristic absorption spectra. We conducted a rapid reaction kinetic analysis of the full-length yeast enzyme in the forward and reverse directions. In the forward direction, we observed formation of the external aldimine of serine (14 mM-1 S-1) and the aminoacrylate intermediate (15 s-1). Homocysteine binds to the aminoacrylate with a bimolecular rate constant of 35 mM-1 s-1 and rapidly converts to cystathionine (180 s-1), leading to the accumulation of a 420 nm absorbing species, which has been assigned as the external aldimine of cystathionine. Release of cystathionine is slow (κ = 2.3 s-1), which is similar to κcat (1.7 s-1) at 15 °C, consistent with this being a rate-determining step. In the reverse direction, cystathionine binds to the enzyme with a bimolecular rate constant of 1.5 mM-1 s-1 and is rapidly converted to the aminoacrylate without accumulation of the external aldimine. The kinetic behavior of the full-length enzyme shows notable differences from that reported for a truncated form of the enzyme lacking the C-terminal third of the protein (Jhee, K. H., Niks, D., McPhie, P., Dunn, M. F., and Miles, E. W. (2001) Biochemistry 40, 10873-10880).