Use of phospholipase D to alter the surface charge of membranes and its effect on the enzymatic activity of D-beta-hydroxybutyrate dehydrogenase.

The effect of an electrostatic potential on the enzymatic activity of D-beta-hydroxybutyrate dehydrogenase was examined. Phospholipase D was used to increase the surface charge and concomitantly the electrostatic potential of submitochondrial membranes. The apparent Km for the negatively charged substrates of D-beta-hydroxybutyrate dehydrogenase increased as the membranes were reacted with phospholipase D. There was a 10-fold increase in the apparent Km for NADH when the content of acidic phospholipids was increased by 24%. The addition of monovalent or divalent cations, which reduced the electrostatic potential, largely reversed the apparent Km changes. At the same ionic strength, divalent cations had a substantially larger effect than monovalent cations. Similar results were obtained when the purified apoenzyme was reconstituted in unilamellar vesicles containing different ratios of phosphatidylcholine and acidic phospholipids. When the apoenzyme was reconstituted into phosphatidylcholine vesicles containing increasing amounts of phosphatidylethanolamine, the apparent Km also increased but to a smaller extent, and increasing the ionic strength did not reverse this effect. The results show that the apparent Km of D-beta-hydroxybutyrate dehydrogenase can be significantly altered by an electrostatic potential as well as other properties of the phospholipid polar head group.