Aldehyde dehydrogenase from human liver

Abstract

Analysis of CNBr fragments and other peptides from human liver cytoplasmic aldehyde dehydrogenase enabled determination of the complete primary structure of this protein. The monomer has an acylated amino terminus and is composed of 500 amino acid residues, including I1 cysteine residues. N o evidence of any microheterogeneity was obtained, supporting the concept that the enzyme is a homotetramer. The disulfiram-sensitive thiol in the protein, earlier identified through its reaction with iodoacetamide, is contributed by a cysteine residue at position 302, while the cysteine which in horse liver mitochondrial aldehyde dehydrogenase is reactive with coenzyme analogs appears to correspond to either Cys-455 or Cys-463. Analysis of glycine distribution and prediction of secondary structures to localize PEP regions typical for coenzyme-binding are not fully unambiguous, but suggest a short region around position 245 as a likely segment for this function. In this region, sequence similarities to parts of a bacterial aspartate-P-semialdehyde dehydrogenase and a mammalian alcohol dehydrogenase were noted. Otherwise, no extensive similarities were detected in comparisons with characterized mammalian enzymes of similar activity or subunit size as aldehyde dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase and glutamate dehydrogenase, respectively).Aldehyde dehydrogenases have a wide substrate specificity and participate in ethanol metabolism through clearance of acetaldehyde. In human liver, two major isoenzymes of aldehyde dehydrogenase are present and have been ascribed to the cytoplasm and mitochondria, as in the livers of other mammalian species [ 1 -51. Structurally, the isoenzymes behave like different proteins in the sense that hybrid molecules are not (but see [6]) usually found [7]. Furthermore, a comparison of short segments from horse cytoplasmic and mitochondrial aldehyde dehydrogenases indicated a high extent of substitutions (about 50%) between the two forms [8]. Immunologically, however, they appear to be homologous [5,9] and the two forms are also similar in many enzymic properties. One notable exception is the much greater sensitivity of the cytoplasmic isoenzyme to inactivation by disulfiram [I, 5, 10, 111. This compound is also used clinically for alcohol aversion therapy, acting through the effects of acetaldehyde accumulation [12].Only recently have detailed molecular aspects of aldehyde dehydrogenase been studied. In the human cytoplasmic isoenzyme, a cysteine-containing segment has been implicated in the reaction with disulfiram because reaction of one of its cysteine residues with iodoacetamide is blocked by disulfiram [I 31. Another segment from horse mitochondrial aldehyde dehydrogenase, also containing a reactive cysteine residue, has been implicated in NAD(H) binding from its reaction with a coenzyme analog [14]. Although not finally proven, the possi- bility of a position close to the active site for one of these cysteine residues appears likely [I 31; this would be compatible with the ...

The cytoplasmic isoenzyme of horse liver aldehyde dehydrogenase

“…text) that have been correlated with positions close to the active site are indicated by vertical arrows predictions for the horse isoenzyme according to Chou and Fasman [17] are also shown in Fig. 2 and differ in only four regions from the predicted secondary structures of the same human isoenzyme [6]. One is the 8-pleated sheet region predicted between positions 122 -133 in the human isoenzyme which is extended to residue 116 and, less certainly, around Gly-123 in the horse isoenzyme.…”

“…The NAD-binding structure is well known in other, shorter dehydrogenases [20,21], and such a domain is likely to be similar in aldehyde dehydrogenase. A comparison of the primary structure of human liver aldehyde dehydrogenase with that of the dehydrogenases of known secondary structure shows only one weak homology region with horse liver alcohol dehydrogenase [6]. That homology region in both enzymes, although not supported by secondary structure predictions in the aldehyde dehydrogenase [6], is part of the coenzyme binding domain in alcohol dehydrogenase and contains some residues important for the binding of NAD (residues Phe-198, Gly-199, Gly-201, Val-222, Asp-223, Ile-224, and Asn-225 in horse liver alcohol dehydrogenase [21]).…”

“…This highly exchanged part is preceded in the primary structure by the most conserved stretch, covering 68 residues between positions 207 -274. Secondary structure .. -- The established human liver enzyme [6] is shown by continuous letters (upper line) whereas for the horse protein (lower line of the two in each pair), a letter is shown only at positions corresponding to proved differences, a dash shows a proved identity, a point a tentative identity, and a comma a tentative difference. Results of secondary structure predictions for the horse protein are also given, and are shown as boxes downwards for fl-pleated sheets, boxes upwards for a-helices and angled signs for reverse turns.…”

“…Regions around functionally active residues have been identified [3, 41 and other short regions have permitted limited comparisons of corresponding sequences to assess both isoenzyme divergence and species divergence [5]. As expected, it was then found that the cytoplasmic isoenzymes from two species (man and horse) are more closely related than the two different isoenzymes within one species.In parallel with the determination of the total primary structure of the human cytoplasmic isoenzyme (see preceding paper [6]), work on the corresponding horse liver isoenzyme has been carried out. Comparisons revealed that a close homology exists.…”

“…In parallel with the determination of the total primary structure of the human cytoplasmic isoenzyme (see preceding paper [6]), work on the corresponding horse liver isoenzyme has been carried out. Comparisons revealed that a close homology exists.…”