Journal
BIOCHEMICAL JOURNAL
Volume 449, Issue -, Pages 109-121Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20120731
Keywords
cystathionine beta-synthase (CBS); enzyme activation; homocystinuria; ligand-binding sites; protein kinetic stability
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Funding
- Junta de Andalucia [P11-CTS-7187]
- Spanish Ministry of Science and Innovation [CSD2009-00088]
- American Heart Association [0920079G, 09GRNT2110159]
- National Institutes of Health [HL065217]
- Jerome Lejeune Foundation
- Ramon y Cajal research contract from the Spanish Ministry of Science and Innovation [RYC-2009-04147]
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CBS (cystathionine beta-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.
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