Department of Physical Chemistry, Faculty of Sciences, University of Granada, Granada 18071, Spain.
Biochem J. 2013 Jan 1;449(1):109-21. doi: 10.1042/BJ20120731.
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.
CBS（胱硫醚β-合酶）是一种四聚体酶，在同型半胱氨酸代谢的调节中必不可少，其活性受变构调节剂 SAM（S-腺苷甲硫氨酸）增强。CBS 的错义突变是遗传性 HCU（同型胱氨酸尿症）的主要原因。在本研究中，我们应用一种新的方法，结合量热法、功能测定和动力学建模，为 SAM 对 WT（野生型）CBS 和七种 HCU 致病突变体的稳定性和活性的影响提供结构和能量方面的见解。我们在 C 端调节域中发现了两组具有不同结构和能量特征的 SAM 结合位点：一组可能参与调节域动力学稳定的高亲和力结合位点，以及一组参与酶激活的低亲和力结合位点。我们表明，WT CBS 中的调节域具有较低的动力学稳定性，在许多 HCU 致病突变体中进一步降低。我们提出，SAM 诱导的稳定可能在调节 WT 和突变型 CBS 在体内的稳态水平方面发挥关键作用。我们的策略对于理解具有复杂结构的蛋白质的配体效应及其在人类遗传疾病中的作用以及开发新的药理学策略可能具有重要价值。
