On the kinetic mechanism of phospholipase D from Streptomyces sp. in an emulsion system

The kinetic constants of the transphosphatidylation reaction between a phospholipid (phosphatidylcholine) and a nucleophile (3-dimethylamino-1-propanol), catalyzed by phospholipase D from Streptomyces PMF in a water-ethyl acetate emulsion system, have been determined. The K-m of the phospholipid was 16.6 mM, the K-m for the nucleophile 1.3 M and the catalytic constant 1.5 x 10(5) min(-1) at pH 5.6 and 25 degrees C. The kinetic pattern was consistent with a ping-pong mechanism modified by a hydrolytic branch. Furthermore, by studying the intermediate partitioning between competing accepters, it was found that transphosphatidylation hydrolysis of different phospholipids occurred through the formation of a common intermediate. These results support the view that phospholipase D-catalyzed reactions take place through the formation, as the first step, of a phosphatidyl-enzyme intermediate. Finally, experiments carried out at different concentrations of phospholipase D showed the phenomenon of interfacial saturation by the enzyme, thus suggesting that only the phospholipase D located at the interface of the water-organic solvent emulsion was active.