title: Human cystathionine β-synthase (CBS) contains two classes of binding sites for S-adenosylmethionine (SAM): complex regulation of CBS activity and stability by SAM.
abstract: CBS (cystathionine β-synthase) is a multidomain tetrameric enzyme essential in the regulation of homocysteine metabolism, whose activity is enhanced by the allosteric regulator SAM (S-adenosylmethionine). Missense mutations in CBS are the major cause of inherited HCU (homocystinuria). In the present study we apply a novel approach based on a combination of calorimetric methods, functional assays and kinetic modelling to provide structural and energetic insight into the effects of SAM on the stability and activity of WT (wild-type) CBS and seven HCU-causing mutants. We found two sets of SAM-binding sites in the C-terminal regulatory domain with different structural and energetic features: a high affinity set of two sites, probably involved in kinetic stabilization of the regulatory domain, and a low affinity set of four sites, which are involved in the enzyme activation. We show that the regulatory domain displays a low kinetic stability in WT CBS, which is further decreased in many HCU-causing mutants. We propose that the SAM-induced stabilization may play a key role in modulating steady-state levels of WT and mutant CBS in vivo. Our strategy may be valuable for understanding ligand effects on proteins with a complex architecture and their role in human genetic diseases and for the development of novel pharmacological strategies.
authors.affiliation: Department of Physical Chemistry, Faculty of Sciences, University of Granada, Granada 18071, Spain. angelpey@ugr.es
authors.forename: Angel L
authors.initials: AL
authors.lastname: Pey
authors.forename: Tomas
authors.initials: T
authors.lastname: Majtan
authors.forename: Jose M
authors.initials: JM
authors.lastname: Sanchez-Ruiz
authors.forename: Jan P
authors.initials: JP
authors.lastname: Kraus
mesh_terms: D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics*; D004789:Enzyme Activation / Q000235:genetics / Q000502:physiology; D006712:Homocystinuria / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011485:Protein Binding / Q000235:genetics; D055550:Protein Stability; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000493:pharmacokinetics / Q000502:physiology*
chemical_list: D012436:S-Adenosylmethionine; D003541:Cystathionine beta-Synthase