Bromoacetophenone as an affinity reagent for human liver aldehyde dehydrogenase.

Area of Science:

• Biochemistry
• Enzymology

Background:

• Human liver aldehyde dehydrogenase (ALDH) comprises isozymes E1 and E2, crucial for aldehyde metabolism.
• Understanding ALDH inactivation mechanisms is vital for comprehending enzyme function and potential therapeutic interventions.

Purpose of the Study:

• To investigate the inactivation mechanism of human liver aldehyde dehydrogenase isozymes E1 and E2 by bromoacetophenone.
• To identify the specific site of interaction and the residues involved in bromoacetophenone-mediated inactivation.

Main Methods:

• Enzyme kinetics (steady-state and saturation) were used to study the interaction of bromoacetophenone and chloroacetophenone with ALDH isozymes.
• Enzyme inactivation stoichiometry and peptide mapping (CNBr and tryptic digests) were employed to identify labeled peptides.
• Amino acid analysis was performed on labeled fragments.

Main Results:

• Bromoacetophenone completely and irreversibly inactivated both ALDH isozymes E1 and E2.
• Kinetic studies indicated interaction at a specific site, potentially the active site, with dissociation constants suggesting similar binding processes.
• Stoichiometric analysis revealed that 4 equivalents of bromoacetophenone led to complete loss of dehydrogenase and esterase activities.
• Peptide mapping identified a preferentially labeled peptide, and amino acid analysis indicated reaction with a distinct residue compared to iodoacetamide.

Conclusions:

• Bromoacetophenone acts as a potent inactivator of human liver aldehyde dehydrogenase isozymes E1 and E2.
• The results strongly suggest that bromoacetophenone reacts with a residue within the active site of aldehyde dehydrogenase.
• The identified reaction site differs from that targeted by iodoacetamide, providing insights into the enzyme's active site structure.