PURIFICATION AND CHARACTERIZATION OF A (R)-3-HYDROXYBUTYRATE DEHYDROGENASE DELETION MUTANT - EVIDENCE FOR C-TERMINAL INVOLVEMENT IN ENZYME ACTIVATION BY LECITHIN
Hp. Langston et al., PURIFICATION AND CHARACTERIZATION OF A (R)-3-HYDROXYBUTYRATE DEHYDROGENASE DELETION MUTANT - EVIDENCE FOR C-TERMINAL INVOLVEMENT IN ENZYME ACTIVATION BY LECITHIN, Archives of biochemistry and biophysics, 327(1), 1996, pp. 45-52
(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.