PURIFICATION AND CHARACTERIZATION OF A (R)-3-HYDROXYBUTYRATE DEHYDROGENASE DELETION MUTANT - EVIDENCE FOR C-TERMINAL INVOLVEMENT IN ENZYME ACTIVATION BY LECITHIN

(R)-3-Hydroxybutyrate dehydrogenase (BDH; EC 1,1.1.30) is a lipid-requ iring enzyme with a specific requirement of phosphatidylcholine for op timal function, The purified enzyme, devoid of lipid, can be reactivat ed with soluble lecithin or by insertion into phospholipid vesicles co ntaining lecithin, In order to obtain insight into the mechanism of li pid activation, a C-terminal deletion mutant was constructed which con tained 18 amino acids less than BDH, The purified deletion mutant had low, but detectable catalytic activity in the absence of phospholipid. However, the addition of either soluble lecithin or phospholipid vesi cles containing lecithin had no effect on enzymatic function, Further experiments were conducted to determine if the deletion mutant had als o lost its ability to bind to phospholipid vesicles and natural membra nes, Our findings indicate that the mutant enzyme binds to both liposo mes and rat liver microsomes. These results suggest that the binding o f BDH to the phosphatidylcholine head group is independent of its inte raction with the apolar core of the phospholipid bilayer. (C) 1996 Aca demic Press, Inc.