Using O-( n-alkyl)- N-( N, N′-dimethylethyl)phosphoramidates to investigate the role of Ca 2+ and interfacial binding in a bacterial phospholipase D

Abstract

O-( n-alkyl)- N-( N, N′-dimethylethyl)phosphoramidates ( n=6, 8, and 10; C n PNC) 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 C n PNC 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 diC 4PC (monomer substrate), C n PNC 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 diC 4PC as the substrate and using concentrations of C 10PNC that were below its CMC, the K i for C 10PNC was 0.030±0.003 mM, ∼13-fold less than the K m for substrate. Aggregated substrates showed significant inhibition of PLD by C n PNC, although as the substrate chain length increased, inhibition by a given C n PNC was diminished. With POPC vesicles, the apparent K i for C 10PNC was 0.030 of the apparent K m. 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 Ca 2+. 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.