Enzymology with a spin-labeled phospholipase C: soluble substrate binding by 31P NMR from 0.005 to 11.7 T.
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.
Pu
Mingming
M
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Feng
Jianwen
J
Redfield
Alfred G
AG
Roberts
Mary F
MF
D001665:Binding Sites; D008279:Magnetic Resonance Imaging / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008823:Micelles; D015394:Molecular Structure; D010713:Phosphatidylcholines / Q000737:chemistry*; D054801:Phosphoinositide Phospholipase C / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012995:Solubility; D013050:Spectrometry, Fluorescence / Q000379:methods; D013113:Spin Labels; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry
D008823:Micelles; D010713:Phosphatidylcholines; D013113:Spin Labels; D014867:Water; D054801:Phosphoinositide Phospholipase C