Visualization of PLP-bound intermediates in hemeless variants of human cystathionine β-synthase: evidence that lysine 119 is a general base

Abstract

Cystathionine β-synthase catalyzes the condensation of serine and homocysteine to give cystathionine in a pyridoxal phosphate (PLP)-dependent reaction. The human enzyme contains a single heme per monomer that is bound in an N-terminal 69 amino acid extension that is missing from the otherwise highly homologous yeast enzyme. The heme dominates the UV–visible spectrum and obscures kinetic characterization of the PLP-bound reaction intermediates. In this study, we have engineered a hemeless mutant of human cystathionine β-synthase by deletion of the N-terminal 69 amino acids. The resulting variant displays ∼40% of the activity seen with the wild type enzyme, binds stoichiometric amounts of PLP, and permits spectral characterization of PLP-based intermediates. The enzyme as isolated exhibits an absorption maximum at 412 nm corresponding to a protonated internal aldimine. Addition of serine shifts the λmax to 420 nm (assigned as the external aldimine) with a broad shoulder between 450 and 500 nm (assigned as the aminoacrylate intermediate). Addition of the product, cystathionine, also leads to formation of an external aldimine (420 nm). Homocysteine elicits a red shift (and a decrease in absorption) in the spectrum from 412 to 424 nm and an increase in absorption at 330 nm, presumably due to formation of a dead-end complex. Mutation of K119, the residue that forms the Schiff base, to alanine results in a ∼103-fold decrease in activity, which increases ∼2-fold in the presence of an exogenous base, ethylamine. Spectral shifts (412 → 420 nm) consistent with the formation of external aldimines are observed in the presence of serine or cystathionine, but an aminoacrylate intermediate is not formed at detectable levels. These results are consistent with an additional role for K119 as a general base in the reaction catalyzed by human cystathionine β-synthase.