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 th e catalytic constant 1.5 x 10(5) min(-1) at pH 5.6 and 25 degrees C. T he kinetic pattern was consistent with a ping-pong mechanism modified by a hydrolytic branch. Furthermore, by studying the intermediate part itioning between competing accepters, it was found that transphosphati dylation hydrolysis of different phospholipids occurred through the fo rmation 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, exp eriments carried out at different concentrations of phospholipase D sh owed the phenomenon of interfacial saturation by the enzyme, thus sugg esting that only the phospholipase D located at the interface of the w ater-organic solvent emulsion was active.