Catalysis of dehydrogenation of 4-trans-(N,N-dimethylamino)cinnamaldehyde by aldehyde dehydrogenase.

4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) is a chromophoric and fluorogenic substrate of aldehyde dehydrogenase. Fluorescence of DACA is enhanced by binding to aldehyde dehydrogenase in the absence of catalysis both in the presence and absence of the coenzyme analogue 5'AMP. DACA binds to aldehyde dehydrogenase with a dissociation constant of 1-3 microM and stoichiometry of 2 mol mol(-1) enzyme. Incorporation of DACA during catalysis was also investigated and found to be 2 mol DACA mol(-1) enzyme. Effect of pH on the stoichiometry of DACA incorporation during catalysis has shown that DACA incorporation remained constant at 2 mol DACA mol(-1) enzyme, despite a 74-fold velocity enhancement between pH 5.0 and 9.0. Increase of pH increased decomposition of enzyme-acyl intermediate without affecting the rate-limiting step of the reaction. At pH 7.0 the pH stimulated velocity enhancement was 10-fold over that at pH 5.0; further velocity enhancement (11.5-fold that of pH 7.0) was achieved by 150 microM Mg(2+) ions. The velocity at pH 7.0 with Mg(2+) exceeded that of pH 9.0, and that at maximal pH stimulation at pH 9.5. It was observed that level of intermediate decreased to about 1 mol mol(-1) enzyme, indicating that Mg(2+) ions increased the rate of decomposition of the enzyme-acyl intermediate and shifted the rate-limiting step of the reaction to another step in the reaction sequence.

Mechanism of inhibition of aldehyde dehydrogenase by citral, a retinoid antagonist.

Low concentrations of citral (3,7-dimethyl-2,6-octadienal), an inhibitor of retinoic acid biosynthesis, inhibited E1, E2 and E3 isozymes of human aldehyde dehydrogenase (EC1.2.1.3). The inhibition was reversible on dilution and upon long incubation in the presence of NAD+; it occurred with simultaneous formation of NADH and of geranic acid. Thus, citral is an inhibitor and also a substrate. Km values for citral were 4 microM for E1, 1 microM for E2 and 0.1 microM for E3; Vmax values were highest for E1 (73 nmol x min-1 x mg-1), intermediate for E2 (17 nmol x min-1 x mg-1) and lowest (0.07 nmol x min-1 x mg-1) for the E3 isozyme. Citral is a 1 : 2 mixture of isomers: cis isomer neral and trans isomer, geranial; the latter structurally resembles physiologically important retinoids. Both were utilized by all three isozymes; a preference for the trans isomer, geranial, was observed by HPLC and by enzyme kinetics. With the E1 isozyme, both geranial and neral, and with the E2 isozyme, only neral obeyed Michaelis-Menten kinetics. With the E2 isozyme and geranial sigmoidal saturation curves were observed with S0.5 of approximately 50 nM; the n-values of 2-2.5 indicated positive cooperativity. Geranial was a better substrate and a better inhibitor than neral. The low Vmax, which appeared to be controlled by either the slow formation, or decomposition via the hydride transfer, of the thiohemiacetal reaction intermediate, makes citral an excellent inhibitor whose selectivity is enhanced by low Km values. The Vmax for citral with the E1 isozyme was higher than those of the E2 and E3 isozymes which explains its fast recovery following inhibition by citral and suggests that E1 may be the enzyme involved in vivo citral metabolism.

Aldehyde dehydrogenase. Covalent intermediate in aldehyde dehydrogenation and ester hydrolysis.

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.

Modification of aldehyde dehydrogenase with dicyclohexylcarbodiimide: separation of dehydrogenase from esterase activity.

Dehydrogenase activity of the cytoplasmic (E1) isozyme of human liver aldehyde dehydrogenase (EC 1.2.1.3) was almost totally abolished (3% activity remaining) by preincubation with dicyclohexylcarbodiimide (DCC), while esterase activity with p-nitrophenyl acetate as substrate remained intact. The esterase reaction of the modified enzyme exhibited a hysteretic burst prior to achieving steady-state velocity; addition of NAD+ abolished the burst. The Km for p-nitrophenyl acetate was increased, but physicochemical properties remained unchanged. The selective inactivation of dehydrogenase activity was the result of covalent bond formation. Protection by NAD+ and chloral, saturation kinetics, and the stoichiometry and specificity of interaction indicated that the reaction of DCC occurred at the active site of the E1 isozyme. The results suggested the some amino acid other than aspartate or glutamate, possibly a cysteine residue, located on a large tryptic peptide of the E1 enzyme, may have reacted with DCC.

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.

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.

Active site of human liver aldehyde dehydrogenase.

Bromoacetophenone (2-bromo-1-phenylethanone) functions as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) and has been found specifically to label a unique tryptic peptide in the enzyme. Amino-terminal sequence analysis of the labeled peptide after purification by two different procedures revealed the following sequence: Val-Thr-Leu-Glu-Leu-Gly-Gly-Lys. Radioactivity was found to be associated with the glutamate residue, which was identified as Glu-268 by reference to the known amino acid sequence. This paper constitutes the first identification of an active site of aldehyde dehydrogenase.