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Aldehyde dehydrogenase. Covalent intermediate in aldehyde dehydrogenation and ester hydrolysis
E E Blatter1, D P Abriola, R Pietruszko
1Center of Alcohol Studies, Rutgers University, Piscataway, NJ 08855-0969.
The Biochemical Journal
|March 1, 1992
Summary
Human aldehyde dehydrogenase enzymes form covalent intermediates with 4-trans-(NN-dimethylamino)cinnamaldehyde (DACA) and 4-trans-(NN-dimethylamino)cinnamoylimidazole (DACI). Cysteine-302 is identified as the key residue forming these intermediates with both aldehyde and ester substrates.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Human aldehyde dehydrogenase (EC 1.2.1.3) plays a crucial role in cellular metabolism.
- Understanding the substrate specificity and active site of aldehyde dehydrogenases is vital for drug development and disease research.
- Previous studies indicated that 4-trans-(NN-dimethylamino)cinnamaldehyde (DACA) and 4-trans-(NN-dimethylamino)cinnamoylimidazole (DACI) are substrates forming chromophoric covalent intermediates.
Purpose of the Study:
- To compare the spectra of covalent intermediates formed by cytoplasmic (E1) and mitochondrial (E2) isoenzymes of human aldehyde dehydrogenase with DACA and DACI.
- To identify the specific amino acid residue involved in the formation of these covalent intermediates.
- To elucidate the mechanism of covalent intermediate formation with different substrate types.
Main Methods:
- Spectroscopic analysis of covalent intermediates formed from DACA and DACI with E1 and E2 isoenzymes.
- Pronase and trypsin digestion of 3H-labelled covalent intermediates followed by peptide purification using ion-exchange and reverse-phase chromatography.
- Amino acid sequencing and identification of labelled residues, including experiments with iodoacetamide-labelled E1 isoenzyme.
Main Results:
- Spectra of covalent intermediates were similar across both substrates (DACA, DACI) and isoenzymes (E1, E2), resembling 4-trnas-(NN-dimethylamino)cinnamoyl-N-acetylcysteine.
- Cysteine was identified as the labelled amino acid in digested covalent intermediates.
- Amino acid sequence analysis pinpointed residues 273-307 as the labelled peptide, with specific labeling at cysteine-302, which is conserved across all sequenced aldehyde dehydrogenases.
Conclusions:
- Cysteine-302 is the active site residue responsible for forming covalent intermediates with both aldehyde (DACA) and ester (DACI) substrates of human aldehyde dehydrogenase.
- The conserved nature of cysteine-302 highlights its critical role in the catalytic mechanism of aldehyde dehydrogenases.
- These findings provide a deeper understanding of aldehyde dehydrogenase enzyme mechanisms and substrate interactions.