Correlation of loss of activity of human aldehyde dehydrogenase with reaction of bromoacetophenone with glutamic acid-268 and cysteine-302 residues. Partial-sites reactivity of aldehyde dehydrogenase
| Autor: | Regina Pietruszko, Darryl P. Abriola, Alexander D. MacKerell |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
Stereochemistry
Molecular Sequence Data Glutamic Acid Aldehyde dehydrogenase Biochemistry Residue (chemistry) Affinity Reagent Glutamates Humans Trypsin Amino Acid Sequence Cysteine Molecular Biology Chromatography High Pressure Liquid chemistry.chemical_classification biology Acetophenones Affinity Labels Cell Biology Glutamic acid Aldehyde Dehydrogenase Peptide Fragments Enzyme assay Isoenzymes Enzyme chemistry Reagent biology.protein Research Article |
| Zdroj: | Biochemical Journal. 266:179-187 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj2660179 |
| Popis: | Bromoacetophenone (2-bromo-1-phenylethanone) has been characterized as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) [MacKerell, MacWright & Pietruszko (1986) Biochemistry 25, 5182-5189], and has been shown to react specifically with the Glu-268 residue [Abriola, Fields, Stein, MacKerell & Pietruszko (1987) Biochemistry 26, 5679-5684] with an apparent inactivation stoichiometry of two molecules of bromoacetophenone per molecule of enzyme. The specificity of bromoacetophenone for reaction with Glu-268, however, is not absolute, owing to the extreme reactivity of this reagent. When bromo[14C]acetophenone was used to label the human cytoplasmic E1 isoenzyme radioactively and tryptic fragmentation was carried out, peptides besides that containing Glu-268 were found to have reacted with reagent. These peptides were purified by h.p.l.c. and analysed by sequencing and scintillation counting to quantify radioactive label in the material from each cycle of sequencing. Reaction of bromoacetophenone with the aldehyde dehydrogenase molecule during enzyme activity loss occurs with two residues, Glu-268 and Cys-302. The activity loss, however, appears to be proportional to incorporation of label at Glu-268. The large part of incorporation of label at Cys-302 occurs after the activity loss is essentially complete. With both Glu-268 and Cys-302, however, the incorporation of label stops after one molecule of bromoacetophenone has reacted with each residue. Reaction with other residues continues after activity loss is complete. |
| Databáze: | OpenAIRE |
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