Influence of model membrane structure on phospholipase D activity

Abstract

Phospholipase D (PLD) catalyzes the hydrolysis of 1,2-dipalmitoylphosphatidylcholine (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 diffraction (GIXD) and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). Spectroscopic analysis of the phosphate groups provides a quantitative estimation of the hydrolysis efficiency. It was found that the PLD activity depends on the substrate structure and exhibits a maximum in the more disordered liquid-expanded phase. Different mixtures of DPPC and DPPA were investigated by GIXD. Phase separation and the presence of two types of condensed phase domains (DPPC-rich and DPPA-rich) were observed. Higher DPPA concentrations inhibit the hydrolysis reaction. The inhibiting DPPA concentration is a function of the monolayer pressure. The inhibition effect of the hydrolysis product depends on the microstructure of the DPPC-rich domains. The tilt angle in DPPC-rich domains decreases with increasing DPPA amount. Such structural changes could be an indication of essential conformational changes in the head group region, which could therefore reduce the accessibility of the POC bond for a PLD attack.