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An improved purification procedure of human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 is presented. This procedure employs only three chromatographic steps to produce homogeneous E1 and E2 isoenzymes at 60% overall yield. The isoenzymes have been tested for homogeneity by electrophoresis of native and denatured species, specific activity determinations following rechromatography, as well as mapping of tryptic and CNBr fragments. Total SH group analysis has also been done on each isoenzyme. The results show that both isoenzymes are homogeneous. Similarities between E1 and E2 isoenzymes are noted in the mobility of about 40% of tryptic fragments, total SH content, and the mobility of two CNBr fragments. The results also show considerable structural differences between the isoenzymes in that CNBr maps show fragments from E1 and E2 of different molecular weight and about 60% of tryptic fragments migrate to distinct locations. Only one of SH-containing tryptic fragments migrates to the same location in both isoenzymes. E1 and E2 each consist of subunits which migrate as single bands in both sodium dodecyl sulfate (SDS) and urea electrophoresis. While the mobility of E1 and E2 subunits in SDS gels is similar, it is different in urea, showing that subunits of E1 are distinct from those of E2 and that the isoenzymes do not share subunits. Structural similarity between isoenzymes must, therefore, result from sequence similarity within regions of distinct polypeptide chains composing E1 and E2 molecules. The results presented offer a simplified procedure for preparation of the homogeneous isoenzymes; they also suggest that E1 and E2 are products of distinct genes which probably diverged from a common genetic ancestor through gene duplication and compartmentation of the cell.

Two functional regions of liver aldehyde dehydrogenase were characterized before; other structures of homologous parts from isoenzymes have now been determined to obtain further information on the isoenzyme relationships. In a 22-residue region from the horse cytoplasmic and mitochondrial isoenzymes, substitutions occur at 12 positions, including a continuous six-residue portion characterized by non-conservative changes. In contrast, the same structure from the cytoplasmic isoenzyme shows exchanges at only three positions when compared to its counterpart from human cytoplasm. A similar estimate of substitution frequency between species is obtained from a larger sampling at 236 positions. Thus, the isoenzyme difference between aldehyde dehydrogenases from the same species is about five-fold greater than the species difference between corresponding isoenzymes. Hence, the relationship between cytoplasmic and mitochondrial aldehyde dehydrogenases, while recognizable, is distant. This is compatible with the fact that a property such as high sensitivity to disulfiram is a characteristic of only the cytoplasmic isoenzyme.

The primary structure of class III alcohol dehydrogenase (dimeric with chi subunits) from human liver has been determined by peptide analyses. The protein chain is a clearly distinct type of subunit distantly related to those of both human class I and class II alcohol dehydrogenases (with alpha, beta, gamma, and pi subunits, respectively). Disregarding a few gaps, residue differences in the chi protein chain with respect to beta 1 and pi occur at 139 and 140 positions, respectively. Compared to class I, the 373-residue chi structure has an extra residue, Cys after position 60, and two missing ones, the first two residues relative to class I, although the N-terminus is acetylated like that for those enzymes. The chi subunit contains two more tryptophan residues than the class I subunits, accounting for the increased absorbance at 280 nm. There are also four additional acidic and two fewer basic side chains than in the class I beta structure, compatible with the markedly different electrophoretic mobility of the class III enzyme. Residue differences between class III and the other classes occur with nearly equal frequency in the coenzyme-binding and catalytic domains. The similarity in the number of exchanges relative to that of the enzymes of the other two classes supports conclusions that the three classes of alcohol dehydrogenase reflect stages in the development of separate enzymes with distinct functional roles. In spite of the many exchanges, the residues critical to basic functional properties are either completely unchanged--all zinc ligands and space-restricted Gly residues--or partly unchanged--residues at the coenzyme-binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE.

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 11 cysteine residues. No 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 beta alpha beta 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-beta-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).

The E1 isoenzyme of human liver aldehyde dehydrogenase (Km acetaldehyde = 30uM, pH 7.0), when incubated with disulfiram at a stoichiometry of four moles disulfiram/tetrameric E1, is immediately inhibited to within 10% of control activity. The inhibition is reversed by 0.1% (v/v) mercaptoethanol, indicating disulfide bridge formation. An indirect attempt to locate, on maps, a peptide binding disulfiram has yielded inconsistent results. Iodoacetamide inhibits E1 slowly; inhibition is facilitated in the presence of NAD, resulting in loss of ca. 90% of control activity. Incubation with 14C iodoacetamide labels a 16,000 dalton CNBr peptide, and a ca. 4,000 dalton tryptic cleavage product. These fragments can be equated with those which have been suggested by disulfiram.

Peptides from rat liver aldehyde dehydrogenase (AIDH) induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment match the AIDH structure from HTC rat hepatoma cells (HTC-AIDH) at all positions examined, indicating induction of the same gene product by two independent routes. This 452 amino acid residue, class 3 AIDH structure differs substantially from the 500-residue AIDH structures isolated from normal liver cytosol (class 1) and mitochondria (class 2). Despite a 29.8% identity in 429 overlapping amino acids vs the human class 1 enzyme (27.7% vs class 2), neither the N- nor C-termini coincide, and gaps are introduced to optimize the alignment. Two residues placed in the active site of human liver AIDH by chemical modification, Cys-302 and Glu-268, are conserved in class 3 AIDH as Cys-243 and Glu-209. Cys-243/302 is the only cysteine residue conserved in all known AIDH structures. Gly-245 and Gly-250 of class 1/2 AIDHs, fitting the patterns of glycine residues in coenzyme binding fold of other dehydrogenases, are also conserved. Otherwise, Cys-49, Cys-162, and Glu-487, to which functional importance has also been ascribed, are not retained in the class 3 structure. Overall, a high conservation of Gly, Pro, and Trp and similar patterns of predicted secondary structure indicate general conservation of tertiary structure, as noted with other distantly related proteins. Three exon boundaries from the human liver mitochondria AIDH gene directly correspond to the N-terminus of the rat class 3 protein and to two of the gaps in the alignment.(ABSTRACT TRUNCATED AT 250 WORDS)

Human red cell aldehyde dehydrogenase (ALDH) resembles the liver cytosolic isozyme in numerous physicochemical properties. This study was undertaken to establish the structural relationship between the erythrocyte and liver ALDH isozymes. The purified red cell ALDH was S-(14C)-carboxymethylated, and cleaved with trypsin. The tryptic digest was fractionated using Sephadex and reversed-phase chromatography. All peptides analyzed were identified within the liver cytosolic enzyme structure. In each case the sequence obtained corresponds exactly to a segment from the human liver cytosolic ALDH. Thus, the erythrocyte enzyme, by virtue of its chemical and structural identity with the liver cytosolic enzyme, may serve as a suitable peripheral enzyme model to understand the cause and mechanism of alcohol abuse-related changes in liver cytosolic ALDH that has been found to be reduced in alcoholics.

A single cysteine residue is selectively alkylated by iodoacetamide in cytoplasmic human liver aldehyde dehydrogenase (isoenzyme E1). The amino acid sequence of a 35-residue fragment containing this residue is determined, showing two additional cysteine residues and also three histidine residues. The alkylation is selective for Cys-30 of this fragment, with only little alkylation even at an adjacent residue, Cys-29. The region examined is likely to be of significance in the reaction of this isoenzyme with disulfiram since disulfiram blocks the selective alkylation.

Two allelic variant alcohol dehydrogenase isoenzymes, beta 2 beta 2-Bern and beta 1 beta 1, coded by the ADH2 locus, were isolated from human livers of Caucasian origin. They represent the "atypical" and "typical" phenotype, respectively. beta 2 beta 2-Bern has a higher specific activity and a lower pH-optimum, has a higher kM for NAD+, is less susceptible to inactivation by iodoacetate, and cannot be activated with chloride ions. In order to define the structural basis for these properties, we determined the amino acid sequence difference between the beta 2-Bern and the beta 1 polypeptide chains. Peptides were prepared by cleavages with trypsin and CNBr, and were purified by exclusion chromatography and reverse phase high performance liquid chromatography. The structural analysis showed that beta 2-Bern differs at only one position from beta 1: Arg-47 in beta 1 is substituted for His-47 in beta 2-Bern. This exchange, which is identical to that reported for the beta 2-Oriental chain, alters the binding of the pyrophosphate group of the coenzyme NAD(H), and also that of iodoacetate, thus explaining the observed differences between beta 2 beta 2-Bern and beta 1 beta 1.

The alpha subunit of human liver alcohol dehydrogenase has been submitted to structural analysis. Together with earlier work on the beta and gamma subunits, the results allow conclusions on the relationship of all known forms of the class I type of the enzyme. Two segments of the alpha subunit were determined; one was also reinvestigated in the beta and gamma subunits. The results establish 11 residue replacements among class I subunits in the segments analyzed and show that the alpha, beta, and gamma protein chains each are structurally distinct in the active site regions, where replacements affect positions influencing coenzyme binding (position 47; Gly in alpha, Arg in beta and gamma) and substrate specificity (position 48; Thr in alpha and beta, Ser in gamma). Residue 128, previously not detected in beta and gamma subunits, corresponds to a position of another isozyme difference (Arg in beta and gamma, Ser in alpha). The many amino acid replacements in alcohol dehydrogenases even at their active sites illustrate that in judgements of enzyme functions absolute importance of single residues should not be overemphasized. Available data suggest that alpha and gamma are the more dissimilar forms within the family of the three class I subunits that have resulted from two gene duplications. The class distinction of alcohol dehydrogenases previously suggested from enzymatic, electrophoretic, and immunological properties therefore also holds true in relation to their structures.

The "atypical' human liver alcohol dehydrogenase dimer, homogeneous for beta 2-Bern chains, was isolated from human liver of Caucasian individuals. It is derived from an allelic variant at the ADH2 gene locus and exhibits a considerably higher specific activity and lower pH optimum than its "typical' counterpart (isoenzyme beta 1 beta 1) from the beta 1-chain predominant in Caucasians. Peptides were prepared by trypsin or CNBr cleavage, and were purified by exclusion chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Structural analysis of the peptides showed that beta 2-Bern differs at one position from beta 1. Thus, Arg-47 in beta 1 is substituted by His in beta 2-Bern. This exchange, compatible with a one-base mutation, explains all functional differences by altered interactions with the pyrophosphate moiety of the coenzyme. The difference is also structurally identical to that found for another atypical beta 2-subunit, the beta 2-Oriental type of major Asian occurrence, linking these two atypical forms of human alcohol dehydrogenase.

Determination of the amino acid sequence of the beta 1 subunit from the class I (pyrazole-sensitive) human liver alcohol dehydrogenase isoenzyme beta 1 beta 1 revealed a 373-residue structure differing at 48 positions (including a gap) from that of the subunit of the well studied horse liver alcohol dehydrogenase EE isoenzyme. The structure deduced is compatible with known differences in composition, ultraviolet absorbance, electrophoretic mobility and catalytic properties between the horse and human enzymes. All zinc-liganding residues of the horse E subunit are strictly conserved in the human beta 1 subunit, despite an earlier report of a mutation involving Cys-46. This residue therefore remains conserved in all known alcohol dehydrogenase structures. However, the total cysteine content of the beta 1 structure is raised from 14 in the subunit of the horse enzyme to 15 by a Tyr----Cys exchange. Most exchanges are on the surface of the molecule and of a well conserved nature. Substitutions close to the catalytic centre are of interest to explain the altered substrate specificity and different catalytic activity of the beta 1 homodimer. Functionally, a Ser----Thr exchange at position 48 appears to be of special importance, since Thr-48 in beta 1 instead of Ser-48 in the horse enzyme can restrict available space. Four other substitutions also line the active-site pocket, and appear to constitute partly compensated exchanges.

Two isoenzymes of alcohol dehydrogenase have been purified from human stomach and characterized with regard to electrophoretic mobility and kinetic properties with ethanol, hexanol, and acetaldehyde. Both undergo a time-dependent formation of multiple electrophoretic bands; the total amount of alcohol dehydrogenase activity in an average human stomach is only about 0.2% of that of the liver.

The structural divergence between the cytoplasmic isoenzymes of aldehyde dehydrogenase from different species was investigated by analysis of peptides from the horse protein, and correlation of the results with the complete primary structure of the human isoenzyme. The amino acid sequences of these two proteins show a high degree of homology (91% of residues compared are identical). The differences observed are spread over the entire polypeptide chains, with only one cluster, which is close to a reactive cysteine residue and also adjacent to the most conserved region (covering 68 residues) in the primary structures of the whole enzymes. The secondary structure predicted for the human isoenzyme is mainly unaffected by the residue differences in the horse isoenzyme, although limited conformational changes might be compatible with an unexpected overrepresentation of differences involving isoleucine (12 of 43 exchanges represent a loss of Ile in the horse protein). Two cysteine residues that correlate with catalytic activity are identically positioned in the enzyme from the two species.

The homodimeric Oriental beta 2 beta 2 isozyme of human liver alcohol dehydrogenase, corresponding to an allelic variant at the ADH2 gene locus, was studied in order to define the amino acid exchange in relation to the beta 1 beta 1 isozyme, the predominant allelic form among Caucasians. Sequence analysis reveals that the amino acid substitution occurs at position 7 of the largest CNBr fragment, corresponding to position 47 of the whole protein chain. Here, the beta 2 form has a histidine residue, while, in common with other characterized mammalian liver alcohol dehydrogenases, the beta 1 form has an arginine residue. This exchange does not affect the adjacent cysteine-46 residue, which is a protein ligand to the active-site zinc atom, thus clarifying previously inconsistent results. The histidine/arginine-47 mutational replacement corresponds to a position that binds the pyrophosphate group of the coenzyme NAD(H); this explains the functional differences between the beta 1 beta 1 and beta 2 beta 2 isozymes, including both a lower pH optimum and higher turnover number of beta 2 beta 2, which is likely to be the mutant form. The exchange demonstrates the existence of parallel but separate mutations in the evolution of alcohol dehydrogenases because these mammalian enzymes differ at exactly the same position by the same type of substitution as is found between a mutant and the wild-type constitutive forms of the corresponding yeast enzyme.