Kinetic evaluation of conversion of phosphatidylcholine to phosphatidylethanolamine by phospholipase D from different sources.
Transphosphatidylation from phosphatidylcholine (PC) to phosphatidylethanolamine (PE) was conducted by seven phospholipase D preparations from different sources, one of which was of cabbage origin and other six of Streptomyces origin. The reactions were carried out at 30 degrees C using mixture of ethyl acetate containing PC and buffer containing ethanolamine and phospholipase D. To obtain the activity ratio of transphosphatidylation and hydrolysis at various ratios of ethanolamine and water concentrations, the apparent rate constant ratios of transphosphatidylation and hydrolysis, (k'3/k3)app, were calculated, keeping the concentration of PC constant (17.8 mM). Among the seven enzymes examined, five showed good transphosphatidylation, in which 100% of PC could be converted to PE at ethanolamine concentrations ranging from 0.3 to 1.0 M, while two showed poor transphosphatidylation activity. At higher ethanolamine concentrations, the reaction rate was decreased due to substrate inhibition. Hydrolytic reactions were conducted at 30 degrees C with respective enzymes by using mixture of ethyl acetate containing PC or PE and buffer containing phospholipase D. The ratio of (kcat/Km)PE/(kcat/Km)PC was calculated to determine the substrate specificity of various phospholipase D enzymes. The values of (k'3/k3)app varied with the origin of the enzymes, whereas the values of (kcat/Km)PE/(kcat/Km)PC were not so different. The results obtained show that (k'3/k3)app is a good parameter to select an enzyme and that the timing of stopping the reaction is also important to avoid the hydrolysis of the product.
Juneja
L R
LR
Department of Food Science and Technology, School of Agriculture, Nagoya University, Japan.
Kazuoka
T
T
Yamane
T
T
Shimizu
S
S
D001692:Biological Transport; D006868:Hydrolysis; D007700:Kinetics; D008433:Mathematics; D010713:Phosphatidylcholines / Q000493:pharmacokinetics*; D010714:Phosphatidylethanolamines / Q000493:pharmacokinetics*; D010739:Phospholipase D / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D013045:Species Specificity; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity
D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010740:Phospholipases; D010739:Phospholipase D