Reversible modification of D-beta-hydroxybutyrate dehydrogenase by diamide

Area of Science:

• Biochemistry
• Enzymology
• Lipid-protein interactions

Background:

• D-beta-Hydroxybutyrate dehydrogenase is a lipid-dependent enzyme crucial for energy metabolism.
• The enzyme requires specific phospholipids, particularly lecithin, for its catalytic activity.
• The purified enzyme (apodehydrogenase) is inactive without lipid reconstitution.

Purpose of the Study:

• To investigate the role of cysteine residues in the function of D-beta-Hydroxybutyrate dehydrogenase.
• To elucidate the mechanism of enzyme inactivation and reactivation.
• To determine the effect of specific chemical modifications on enzyme activity and coenzyme binding.

Main Methods:

• Enzyme purification and lipid reconstitution.
• Chemical modification using N-ethylmaleimide and diamide.
• Enzyme activity assays.
• Spectroscopic analysis (fluorescence, resonance energy transfer).

Main Results:

• Only two of six cysteine residues were accessible for modification.
• Diamide treatment inactivated the enzyme by modifying one accessible sulfhydryl group, reversible by dithiothreitol.
• N-ethylmaleimide modification of the other sulfhydryl did not abolish activity.
• Diamide modification prevented coenzyme binding site alterations, affecting NAD(H) binding.

Conclusions:

• A specific sulfhydryl group is critical for D-beta-Hydroxybutyrate dehydrogenase activity.
• Diamide-induced inactivation involves modification of this critical sulfhydryl, impacting the coenzyme binding site.
• The enzyme's structure and function are sensitive to modifications at specific cysteine residues.