title: Yeast cystathionine beta-synthase reacts with L-allothreonine, a non-natural substrate, and L-homocysteine to form a new amino acid, 3-methyl-L-cystathionine.
abstract: Our studies of the reaction mechanism of cystathionine beta-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.
authors.affiliation: Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0830, USA.
authors.forename: Kwang-Hwan
authors.initials: KH
authors.lastname: Jhee
authors.forename: Dimitri
authors.initials: D
authors.lastname: Niks
authors.forename: Peter
authors.initials: P
authors.lastname: McPhie
authors.forename: Michael F
authors.initials: MF
authors.lastname: Dunn
authors.forename: Edith Wilson
authors.initials: EW
authors.lastname: Miles
mesh_terms: D002942:Circular Dichroism; D003540:Cystathionine / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000737:chemistry; D003541:Cystathionine beta-Synthase / Q000378:metabolism*; D006710:Homocysteine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012694:Serine / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D013912:Threonine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: C449840:3-methylcystathionine; D006710:Homocysteine; D013912:Threonine; D003540:Cystathionine; D012694:Serine; D011732:Pyridoxal Phosphate; D003541:Cystathionine beta-Synthase