Reaction Mechanism of the Trinuclear Zinc Enzyme Phospholipase C: A Density Functional Theory Study

Phosphatidylcholine-preferring phospholipase C is a trinuclear zinc-dependent phosphodiesterase, catalyzing the hydrolysis of choline phospholipids. In the present study, density functional theory is used to investigate the reaction mechanism of this enzyme. Two possible mechanistic scenarios were considered with a model of the active site designed on the basis of the high resolution X-ray crystal structure of the native enzyme. The calculations show that a Zn1 and Zn3 bridging hydroxide rather than a Zn1 coordinated water molecule performs the nucleophilic attack on the phosphorus center. Simultaneously, Zn2 activates a water molecule to protonate the leaving group. In the following step, the newly generated Zn2 bound hydroxide makes the reverse attack, resulting in the regeneration of the bridging hydroxide. The first step is calculated to be rate-limiting with a barrier of 17.3 kcal/mol, in good agreement with experimental kinetic studies. The zinc ions are suggested to orient the substrate for nucleophilic attack and provide electrostatic stabilization to the dianionic penta-coordinated trigonal bipyramidal transition states, thereby lowering the barrier.