Influence of model membrane structure on phospholipase D activity

Phospholipase D (PLD) catalyzes the hydrolysis of 1,2-dipalmitoylphosphatid ylcholine (DPPC) to 1,2-dipalmitoylphosphatidic acid (DPPA). The influence of substrate (DPPC) structure on the efficiency and rate of the hydrolysis reaction has been investigated in monolayers by grazing incidence X-ray dif fraction (GIXD) and polarization-modulated infrared reflection absorption s pectroscopy (PM-IRRAS). Spectroscopic analysis of the phosphate groups prov ides a quantitative estimation of the hydrolysis efficiency. It was found t hat the PLD activity depends on the substrate structure and exhibits a maxi mum in the more disordered liquid-expanded phase. Different mixtures of DPP C and DPPA were investigated by GIXD. Phase separation and the presence of two types of condensed phase domains (DPPC-rich and DPPA-rich) were observe d. Higher DPPA concentrations inhibit the hydrolysis reaction. The inhibiti ng DPPA concentration is a function of the monolayer pressure. The inhibiti on effect of the hydrolysis product depends on the microstructure of the DP PC-rich domains. The tilt angle in DPPC-rich domains decreases with increas ing DPPA amount. Such structural changes could be an indication of essentia l conformational changes in the head group region, which could therefore re duce the accessibility of the POC bond for a PLD attack.