Blatter E E, Abriola D P, Pietruszko R
Center of Alcohol Studies, Rutgers University, Piscataway, NJ 08855-0969.
Biochem J. 1992 Mar 1;282 ( Pt 2)(Pt 2):353-60. doi: 10.1042/bj2820353.
4-trans-(NN-Dimethylamino)cinnamaldehyde (an aldehyde, DACA) and 4-trans-(NN-dimethylamino)cinnamoylimidazole (an amide, DACI) have been shown to be substrates for human aldehyde dehydrogenase (EC 1.2.1.3) which form chromophoric covalent intermediates. The spectra of covalent intermediates from both the cytoplasmic (E1) and mitochondrial (E2) isoenzymes derived from DACA and DACI were compared. The spectra were similar when either substrate was used, and also when the two isoenzymes were compared, and resembled that obtained for 4-trnas-(NN-dimethylamino)cinnamoyl-N-acetylcysteine, but differed from the spectrum of 4-trans-(NN-dimethylamino)cinnamoyl ethyl ester. After extensive digestion of the covalent intermediates from both 3H-labelled DACA and DACI with Pronase and purification, the labelled amino acid was identified as cysteine. Covalent intermediates from both DACA and DACI were also digested with trypsin, and labelled peptides were purified by ion-exchange and reverse-phase chromatography. Amino acid sequence analysis showed that the peptide comprising residues 273-307 was labelled by both DACA and DACI. The radioactive label at cysteine residues 301-303 of the primary structure could be unequivocally identified by employing the DACA derivative. Assignment of label to cysteine-302 was achieved by employing iodoacetamide-labelled E1 isoenzyme (iodoacetamide specifically labels cysteine-302), in which case there was no formation of the covalent intermediate from either DACA or DACI. In addition, cysteine-302 is the only cysteine residue conserved in all aldehyde dehydrogenases sequenced. Thus cysteine-302 is the amino acid residue that forms a covalent intermediate with both aldehyde and ester substrates.
4-反式-(N,N-二甲基氨基)肉桂醛（一种醛，DACA）和4-反式-(N,N-二甲基氨基)肉桂酰咪唑（一种酰胺，DACI）已被证明是人类醛脱氢酶（EC 1.2.1.3）的底物，它们会形成发色共价中间体。比较了来自DACA和DACI的细胞质（E1）和线粒体（E2）同工酶的共价中间体的光谱。当使用任何一种底物时，以及比较这两种同工酶时，光谱都是相似的，并且类似于从4-反式-(N,N-二甲基氨基)肉桂酰-N-乙酰半胱氨酸获得的光谱，但与4-反式-(N,N-二甲基氨基)肉桂酰乙酯的光谱不同。用链霉蛋白酶对来自3H标记的DACA和DACI的共价中间体进行广泛消化并纯化后，鉴定出标记的氨基酸为半胱氨酸。来自DACA和DACI的共价中间体也用胰蛋白酶消化，标记的肽通过离子交换和反相色谱法纯化。氨基酸序列分析表明，由273 - 307位残基组成的肽被DACA和DACI都标记了。通过使用DACA衍生物可以明确鉴定一级结构中半胱氨酸残基301 - 303处的放射性标记。通过使用碘乙酰胺标记的E1同工酶（碘乙酰胺特异性标记半胱氨酸-302）实现了将标记分配到半胱氨酸-302，在这种情况下，DACA或DACI都不会形成共价中间体。此外，半胱氨酸-302是所有已测序醛脱氢酶中唯一保守的半胱氨酸残基。因此，半胱氨酸-302是与醛和酯底物都形成共价中间体的氨基酸残基。
