دراسة تفاعلات الاستبدال الإنزيمي لمشتقات الفوسفاتيديل في الوسط الكحولي والحامضي

Study of Enzymatic Substitution Reactions of Phosphatidyl Derivative with Alcohol and Acidic Medium

ABSTRACT

The phospholipids (PLs) play an important role in building up all the cells membrane of the animals. In addition, PLs are amphiphilic compounds that are widely found in the living system. In general, PLs can be divided into two groups: sphingomyelin and glycerophospholipids, which include phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol and phosphatidylinositol.
The hydrolysis of the PLs is the main reaction for lipolysis that catalyzes in presence of specific enzymes known as phospholipases. In vitro, there are many studies to understand the mechanism of the phospholipolytic PLs and what the effect of the environment on this reaction.
The current study focused on monitoring of the hydrolysis reaction of PC in acidic medium and compared of the same reaction in alcoholic medium to find the effect of alcoholic environment on this reaction. In addition, this work focuses on the enzymatic hydrolysis reactions of the two PLs derivatives, which are phosphatidylcholine and phosphatidylserine, in water as well as in ethanol catalyzing by phospholipase D to find the impact of the toxic concentration of ethanol that may lead to damage to the cell membrane. The study shows that there is clear impact of the change of the nitrogen bases of phospholipids and the environment of the hydrolysis on the activity of the enzymatic catalyzing.
All products for the hydrolysis of PC in both the acidic and alcoholic environments were isolated and characterized using the GC-Mass, NMR, and FTIR spectroscopy.
Depending on the results, the mechanisms of the hydrolysis of PC in acidic and alcoholic environments were suggested. The final products of the hydrolysis of PC in acidic medium were choline chloride, palmitic acid, octadeca-9,12-dienoic acid, and glycerol-3-phospate. However, the hydrolysis of PC in ethanol provided the following products: choline chloride, ethyl palmitate, ethyl octadeca-9,12-dienoate, and glycerol-3-ethylphospate. It can be noted that the glycerol-3-phosphate was formed in both reactions as backbone of the PC also the choline chloride was produced during the dissociation from the phosphate group of the PC.
The same reactions above were repeated for two phospholipids, which were the PC and PS, respectively, in presence of phospholipase D (PLD) as catalyst. The kinetics of dissociation of the choline from the phosphate-group in presence of PLD were studied by applying the Michaelis-Menten equation. The maximum velocity (Vmax) and the Michaelis-Menten constant (Km) for all the enzymatic hydrolysis of PC and PS in presence of PLD at the acidic and alcoholic environments were determined. The affinity between the phospholipid PC and the enzyme in the case of using ethanol as a solvent was the least among the other reactions because it had the highest Km value of 3.664. However, the enzymatic hydrolysis reaction of PS was the highest affinity among others by having the lowest Km value.
In addition, the optimization of the enzymatic hydrolysis reactions which were included the concentration of the substrate, the enzymatic activity, the pH, and the temperature were performed to ensure that this enzymatic hydrolysis mimics that occurs in a living system. The thermodynamic parameters, which include enthalpy change (∆H), Gibbs free energy change (∆G) and entropy change (∆S) were determined. All the enzymatic hydrolysis reactions of PC and PS in presence of PLD were thermodynamically favorable and not spontaneous.
This study has exhibited that all hydrolysis reactions of the phospholipids in the absence of the enzyme are slower than that in presence of the PLD. Consequently, all products for the hydrolysis of PC without enzyme were smoothly isolated and characterized. However, it could not isolate the products for the enzymatic hydrolysis of the PC and PS due to the reactions being very fast.
Despite the similarity in mechanisms of the hydrolysis reaction of the phospholipids (PC and PS) in different environments (acidic and alcoholic), the differences in byproducts may provide us with a good vision to predict the impact of the solvent on this process. Due to the various applications of phosphatidylcholine and phosphatidylserine, understanding of the effect of the alcoholic environment can help pharmaceutical, cosmetic, and food factories to design appropriate experiments to reduce the impact of these byproducts on the main product efficiency and yield.