A PROPOSAL FOR THE CATALYTIC MECHANISM IN PHOSPHOLIPASE-C BASED ON INTERACTION ENERGY AND DISTANCE GEOMETRY CALCULATIONS

The non-specific phospholipase C from Bacillus cereus preferentially h ydrolyses phosphatidylcholine but is also active against phosphatidyls erine, phosphatidylethanolamine and at a much lower level, sphingomyel in. A minimal substrate model containing all required structural and c onfigurational elements of a high affinity substrate was docked into t he active site. The enzyme-substrate attachment points were from molec ular interaction energy calculations using the program GRID and from a previous phosphate inhibitor complex structure. Available conformatio nal space for the substrate was sampled by distance geometry calculati ons using the program DGEOM. This investigation clearly identifies the attacking nucleophile, a catalytically favourable orientation of the phosphate group in its tetra-, as well as its penta-, coordinated stat e and a crucial stabilizing environment for the alkoxide intermediate. Based on this information a complete catalytic cycle is proposed.