Bromoacetophenone as an affinity reagent for human liver aldehyde dehydrogenase.

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TitleBromoacetophenone as an affinity reagent for human liver aldehyde dehydrogenase.
Publication TypeJournal Article
Year of Publication1986
AuthorsMackerell, AD, MacWright, RS, Pietruszko, R
JournalBiochemistry
Volume25
Issue18
Pagination5182-9
Date Published1986 Sep 09
ISSN0006-2960
KeywordsAcetophenones, Affinity Labels, Aldehyde Dehydrogenase, Humans, Kinetics, Liver
Abstract

Human liver aldehyde dehydrogenase isozymes E1 and E2 (EC 1.2.1.3) are both completely and irreversibly inactivated by bromoacetophenone (2-bromo-1-phenylethanone). Steady-state kinetics with both acetophenone and chloroacetophenone indicated interaction with the same enzyme form as the aldehyde substrate. Saturation kinetics with chloroacetophenone and bromoacetophenone indicated interaction at a specific site on the enzyme surface and gave a dissociation constant similar to that from steady-state kinetics, suggesting that the same processes were being observed by both methods and that the active site may be involved. Protection against inactivation was afforded by chloral and NAD together. Stoichiometry of inactivation showed the first 2 equiv per tetramer to abolish the majority of catalytic activity; 4 equiv inactivated both isozymes with complete loss of esterase, NAD-stimulated esterase, and dehydrogenase activities. Peptide mapping of enzyme modified with [carbonyl-14C]bromoacetophenone of CNBr digests (E1) and tryptic digests (E1 and E2) showed one peptide to be preferentially labeled. The above results together with the similarity of bromoacetophenone to the substrate benzaldehyde suggest bromoacetophenone may react with a residue in the active site of aldehyde dehydrogenase. Amino acid analysis of the labeled E1 tryptic fragment indicated reaction with a different peptide from that with which iodoacetamide reacts.

Alternate JournalBiochemistry
PubMed ID3768340
Grant ListAA00046 / AA / NIAAA NIH HHS / United States
AA00186 / AA / NIAAA NIH HHS / United States