Crystal structures of phosphate, iodide and iodate-inhibited phospholipase C from Bacillus cereus and structural investigations of the binding of reaction products and a substrate analogue.
The crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and inorganic phosphate (Pi), which is an inhibitor, has been determined and refined to 2.1 A resolution. The final R-factor is 19.7%. We have also studied the binding of two other inhibitors, iodide and iodate, to PLC. X-ray data for these two complexes were collected to 2.8 A resolution during the search for heavy-atom derivatives. A series of screening experiments where PLC crystals have been treated with several reaction products and a substrate analogue were carried out to clarify the question of substrate binding. The results have so far been ambiguous but are discussed briefly. Phosphate and iodate are both found to bind to the three metal ions in the protein molecule, suggesting that these ions are involved directly in the catalytic process and thereby identifying the active site. PLC also binds nine iodide ions, eight of which are on the surface of the molecule and of lower occupancy. The ninth blocks the entrance to the active site cleft and is of higher occupancy. Altogether, these results suggest that the substrate, a phospholipid, is associated directly with the metal ions during catalysis.
Hansen
S
S
Department of Chemistry, University of Tromsø, Norway.
Hansen
L K
LK
Hough
E
E
D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D003460:Crystallization; D007452:Iodates / Q000737:chemistry / Q000378:metabolism*; D007454:Iodides / Q000737:chemistry / Q000378:metabolism*; D046911:Macromolecular Substances; D008958:Models, Molecular; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D014961:X-Ray Diffraction; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D007452:Iodates; D007454:Iodides; D046911:Macromolecular Substances; D010710:Phosphates; D010738:Type C Phospholipases; D015032:Zinc