自旋标记磷脂酶C的酶学研究：通过31P核磁共振在0.005至11.7特斯拉下测定可溶性底物结合情况
Enzymology with a spin-labeled phospholipase C: soluble substrate binding by 31P NMR from 0.005 to 11.7 T.
作者信息
Pu Mingming, Feng Jianwen, Redfield Alfred G, Roberts Mary F
机构信息
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, USA.
出版信息
Biochemistry. 2009 Sep 8;48(35):8282-4. doi: 10.1021/bi901190j.
Abstract
31P NMR relaxation studies from 0.005 to 11.7 T are used to monitor water-soluble inositol 1,2-(cyclic) phosphate (cIP) binding to phosphatidylinositol-specific phospholipase C spin-labeled at H82C, a position near the active site of the enzyme, and to determine how activating phosphatidylcholine (PC) molecules affect this interaction. We show that, in the absence of an interface, cIP binding to the protein is not rate-limiting, and that lower activation by PC vesicles as opposed to micelles is likely due to hindered product release. The methodology is general and could be used for determining distances in other weakly binding small molecule ligand-protein interactions.
摘要
利用从0.005到11.7特斯拉的31P NMR弛豫研究来监测水溶性肌醇1,2 -（环）磷酸酯（cIP）与在靠近酶活性位点的H82C位置自旋标记的磷脂酰肌醇特异性磷脂酶C的结合，并确定激活磷脂酰胆碱（PC）分子如何影响这种相互作用。我们表明，在没有界面的情况下，cIP与蛋白质的结合不是限速步骤，并且与胶束相比，PC囊泡的较低激活可能是由于产物释放受阻。该方法具有通用性，可用于确定其他弱结合小分子配体 - 蛋白质相互作用中的距离。
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