On the Kinetic Mechanism of Phospholipase D from Streptomyces SP. In an Emulsion System

Abstract

The kinetic constants of the transphosphatidylation reaction between a phospholipid (phos phatidylcholine) 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 16.6mM, the K_m for the nucleophile 1.3 M and the catalytic constant 1.5 × 10⁵min⁻¹ at pH 5.6 and 25°C. The kinetic pattern was consistent with a ping-pong mechanism modified by a hydrolytic branch. Furthermore, by studying the intermediate partitioning between competing acceptors, 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 inter-facial saturation by the enzyme, thus suggesting that only the phospholipase D located at the interface of the water-organic solvent emulsion was active.

Key Words:

• Phospholipase D
• Streptomyces
• Transphosphatidylation
• Kinetic constants
• Interfacial saturation
• Emulsion system