Phosphoryl transfers of the phospholipase D superfamily: a quantum mechanical theoretical study.

The HKD-containing Phospholipase D superfamily catalyzes the cleavage of the headgroup of phosphatidylcholine to produce phosphatidic acid and choline. The mechanism of this cleavage process is studied theoretically. The geometric basis of our models is the X-ray crystal structure of the five-coordinate phosphohistidine intermediate from Streptomyces sp . Strain PMF (PDB Code = 1V0Y ). Hybrid ONIOM QM:QM methodology with Density Functional Theory (DFT) and semiempirical PM6 (DFT:PM6) is used to acquire thermodynamic and kinetic data for the initial phosphoryl transfer, subsequent hydrolysis, and finally, the formation of the experimentally observed ″dead-end″ phosphohistidine product (PDB Code = 1V0W ). The model contains nineteen amino acid residues (including the two highly conserved HKD-motifs), four explicit water molecules, and the substrate. Via computations, the persistence of the short-lived five-coordinate phosphorane intermediate on the minutes times scale is rationalized. This five-coordinate phosphohistidine intermediate energetically exists between the hydrolysis event and ″substrate reorganization″ (the reorganization of the in vitro model substrate within the active site). Computations directly support the thermodynamic favorability of the in vitro four-coordinate phosphohistidine product. In vivo, the activation energy of substrate reorganization is too high, perhaps due to a combination of substrate immobility when embedded in the lipid bilayer, as well as its larger steric bulk compared to the compound used in the in vitro substrate soaks. On this longer time scale, the enzyme will migrate along the lipid membrane toward its next substrate target, rather than promote the formation of the dead-end product.