Darryl P. Abriola

Localization of Cysteine 302 at the Active Site of Aldehyde Dehydrogenase

The superreactive cysteine was first identified in human cytoplasmic aldehyde dehydrogenase El isozyme, before its primary structure was known, as a part of 35 residue tryptic peptide (Hempel, 1981; Hempel and Pietruszko, 1981; Hempel et al., 1982) by employing iodoacetamide. When the primary structures of the El and E2 isozymes were established (Hempel et al., 1984, 1985; Hsu et al., 1985), this cysteine was found to occupy position 302 in a 500 amino acid residue polypeptide chain. Iodoacetamide fulfilled all criteria for an aldehyde-competitive, active-site-directed reagent with the exception of total inactivation of the mitochondrial E2 isozyme. Since that time, other investigators have also attempted to identify active site residues. Coenzyme-based affinity reagents (von Bahr-Lindstrom et al., 1985) identified cysteines 369 and 302, Nethylmaleimide identified cysteine 49 and 162 (Tu and Weiner, 1988 a,b) and dimethylaminocinnamaldehyde identified serine 74 (Loomes et al., 1990). Our laboratory developed a substrate-based affinity reagent, bromo-acetophenone (MacKerell et al., 1986), which identified glutamate 268 (Abriola et al., 1987).

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. TheKm 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 that 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.

Aldehyde inhibitors of aldehyde dehydrogenases.

Aldehyde dehydrogenases catalyze aldehyde dehydrogenation as well as ester hydrolysis. Nitrate esters e.g. isosorbide nitrates and nitroglycerin inhibit the enzyme. Mechanism of inhibition which involves aldehyde dehydrogenase catalyzed formation of a reactive species (Mukerjee and Pietruszko, 1994), which inactivates the enzyme via covalent bond formation, was previously discussed (Pietruszko et al., 1995) in this series. In addition, aldehyde dehydrogenases share a common property of being inhibited by aldehyde inhibitors such as chloral, citral, methylglyoxal and 4-dia1ky1amino benzaldehyde, whose structure resembles that of aldehyde substrates (Figure I). It was interesting, therefore, to investigate how these compounds inhibit the enzyme. Methylglyoxal is a natural metabolite which is formed enzymatically and non-enzymatically from triose phosphates and from acetone by cytochrome P-450; it also arises from metabolism of threonine. Because of its chemical reactivity toward proteins and nucleic acids, methylglyoxal is considered to be a toxic compound (Kalapos, 1994; Chaplen et al., 1998); modification of proteins and nucleic acids by methylglyoxal has been postulated to be a possible cause of development of diabetic complications (Vander Jagt et al., 1992). Methylglyoxal is metabolized by glyoxalase which catalyzes the conversion of methyl glyoxal to D-Iactate in the presence of reduced glutathione. Aldose reductase and aldehyde reductase catalyze reduction of methylglyoxal to acetol and D-Iactaldehyde. Another metabolic route for methylglyoxal is its oxidation to pyruvate by α-ketoaldehyde dehydrogenase (Monder, 1967). The structure of methylglyoxal is shown in Figure 1.

Binding and incorporation of 4-trans-(N,N-dimethylamino) cinnamaldehyde by aldehyde dehydrogenase.

Abstract4-trans-(N,N-Dimethylamino)cinnamaldehyde (DACA) is a chromophoric substrate of aldehyde dehydrogenase (EC 1.2.1.3) whose fate can be followed during catalysis. During this investigation we found that DACA also fluoresces and that this fluorescence is enhanced and blue-shifted upon binding to aldehyde dehydrogenase. Binding of DACA to aldehyde dehydrogenase also occurs in the absence of coenzyme. Benzaldehyde (a substrate), acetophenone (a substrate-competitive inhibitor), and the substrate-competitive affinity reagent bromoaceto-phenone interfere with DACA binding. Thus, DACA binds to the active site and can be employed for titration of active aldehyde dehydrogenase. Both E1 and E2 isozymes, which are homotetramers, bind DACA with dissociation constants of 1–4 μM with a stoichiometry of 2 mol DACA/mol enzyme. The stoichiometry of enzyme–acyl intermediate was also found to be 2 mol DACA/mol enzyme for both E1 and E2 isozymes. Thus, both enzymes appear to have only two substrate-binding sites which participate in catalysis. The level of enzyme–acyl intermediate remained constant at different pH values, showing that enhancement of velocity with pH was not due to altered DACA–enzyme levels. When the reaction velocity was increased even further by using 150 μM Mg2+ the intermediate level was decreased, suggesting that both increased pH and Mg2+ promote decomposition of the DACA–enzyme intermediate. Titration with DACA permits study of aldehyde substrate catalysis before central complex interconversion.

Metabolism of Retinaldehyde by Human Liver and Kidney

In mamian organisms retinoids and their derivatives are important in regulation of divershysiological functions. Retinoic acid, the most potent of naturally occurring retinoids, only rntly has been recognized as a major hormone in cell differentiation and development (Gudas994). In mammals biosynthesis of retinoids proceeds via central or excent cleavage of carotene to retinaldehyde followed by its reduction to retinol or oxidation to retic acid (Goodman and Huang, 1965; Olson and Hyashi, 1965; Wang et al., 1996b)