Yeast Cystathionine β-Synthase Reacts with l-Allothreonine, a Non-Natural Substrate, and l-Homocysteine To Form a New Amino Acid, 3-Methyl-l-cystathionine

Our studies of the reaction mechanism of cystathionine β-synthase from yeast (Saccharomyces cerevisiae) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme. The enzyme catalyzes the reaction of l-serine with l-homocysteine to form l-cystathionine through a series of pyridoxal phosphate intermediates. In this work, we explore the substrate specificity of the enzyme by use of substrate analogues combined with kinetic measurements under pre-steady-state conditions and with circular dichroism and fluorescence spectroscopy under steady-state conditions. Our results show that l-allothreonine, but not l-threonine, serves as an effective substrate. l-Allothreonine reacts with the pyridoxal phosphate cofactor to form a stable 3-methyl aminoacrylate intermediate that absorbs maximally at 446 nm. The rapid-scanning stopped-flow results show that the binding of l-allothreonine as the external aldimine is faster than formation of the 3-methyl aminoacrylate intermediate. The 3-methyl aminoacrylate intermediate reacts with l-homocysteine to form a new amino acid, 3-methyl-l-cystathionine, which was characterized by nuclear magnetic resonance spectroscopy. This new amino acid may be a useful analogue of l-cystathionine.