Limited proteolysis of D-beta-hydroxybutyrate dehydrogenase: relationships between phospholipid-protein interactions and catalytical activity.

Different forms of D-beta-hydroxybutyrate dehydrogenase were submitted to various proteases in order to get information on enzyme molecular structure and on phospholipid -enzyme interaction. Except for leucinaminopeptidase, all proteases tested inactivated the phospholipid-free enzyme, while no inactivation was observed with the lecithin-enzyme complex. However, non-reactivating phospholipid gave very poor protection against proteases. After endopeptidase treatment, a new band of 25,000 Mr appeared instead of the 32,000 Mr band (apodehydrogenase). Surprisingly, the so-called protected form of the enzyme (lecithin-complex) was also proteolysed while still enzymatically active. Carboxypeptidase A inactivated quite thoroughly the enzyme although the 32,000 Mr band appeared unaffected. These results demonstrate that the configuration of the phospholipid-free apodehydrogenase is quite vulnerable to most proteases, in contrast to the configuration of the lecithin-complexed enzyme. The N-terminal end is probably blocked while the C-terminal end looks quite important for enzymatic activity.