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 preferentiallyhydrolyses phosphatidylcholine but is also active against phosphatidylserine,phosphatidylethanolaniine and at a much lower level, sphingomyelin.A minimal substrate model containing all required structuraland configurational elements of a high affinity substrate wasdocked into the active site. The enzyme–substrate attachmentpoints were from molecular interaction energy calculations usingthe program GRID and from a previous phosphate inhibitor complexstructure. Available conformational space for the substratewas sampled by distance geometry calculations using the programDGEOM. This investigation clearly identifies the attacking nucleophile,a catalytically favourable orientation of the phosphate groupin its tetra-, as well as its penta-, coordinated state anda crucial stabilizing environment for the alkoxide intermediate.Based on this information a complete catalytic cycle is proposed.