Using O-(n-alkyl)-N-(N,N'-dimethylethyl)phosphoramidates to investigate the role of Ca2+ and interfacial binding in a bacterial phospholipase D.

O-(n-alkyl)-N-(N,N'-dimethylethyl)phosphoramidates (n=6, 8, and 10; CnPNC) were synthesized and characterized as inhibitors of phospholipase D (PLD) activity toward phosphatidylcholine presented as monomers, micelles, and bilayers. Detailed studies with recombinant Streptomyces chromofuscus PLD, a Ca(2+)-activated enzyme that does not show large changes in catalytic activity toward the same substrate as a monomer or micelle, showed that the longer the inhibitor chain length, the more potent CnPNC is as a competitive inhibitor toward all the substrates. However, the physical state of the inhibitor did affect the maximum inhibition attainable. For a fixed concentration of diC4PC (monomer substrate), CnPNC inhibition reached a maximum around the CMC of the inhibitor; the inhibition was reduced at higher inhibitor concentrations, in part caused by the lower solubility of the aggregated inhibitor. With diC4PC as the substrate and using concentrations of C10PNC that were below its CMC, the Ki for C10PNC was 0.030+/-0.003 mM, approximately 13-fold less than the Km for substrate. Aggregated substrates showed significant inhibition of PLD by CnPNC, although as the substrate chain length increased, inhibition by a given CnPNC was diminished. With POPC vesicles, the apparent Ki for C10PNC was 0.030 of the apparent Km. The availability of these inhibitors allowed us to show that PC analogues can bind to the active site of S. chromofuscus PLD in the absence of Ca2+. Once bound at the active site, the inhibitor does not significantly affect the divalent ion-dependent partitioning of the enzyme to PC surfaces. Of the two other PLD enzymes examined, cabbage PLD, but not Streptomyces sp. PMF, was able to catalyze the cleavage of the P-N bond. Differential susceptibility of PLDs to these phosphoramidates may eventually be useful in studying PLD isozymes in cells.