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-requiring enzyme with a specific requirement of phosphatidylcholine for optimal function. The purified enzyme, devoid of lipid, can be reactivated with soluble lecithin or by insertion into phospholipid vesicles containing lecithin. In order to obtain insight into the mechanism of lipid activation, a C-terminal deletion mutant was constructed which contained 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 vesicles containing lecithin had no effect on enzymatic function. Further experiments were conducted to determine if the deletion mutant had also lost its ability to bind to phospholipid vesicles and natural membranes. Our findings indicate that the mutant enzyme binds to both liposomes and rat liver microsomes. These results suggest that the binding of BDH to the phosphatidylcholine head group is independent of its interaction with the apolar core of the phospholipid bilayer.
Langston
H P
HP
Department of Biological Sciences, University of Alabama, Tuscaloosa, 35487, USA.
Jones
L
L
Churchill
S
S
Churchill
P F
PF
D000818:Animals; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008862:Microsomes, Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D016296:Mutagenesis; D010713:Phosphatidylcholines / Q000494:pharmacology*; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence
D008081:Liposomes; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D006884:Hydroxybutyrate Dehydrogenase