Tissue distribution of the five identified classes of human alcohol dehydrogenase was studied by assessment of mRNA levels in 23 adult and four fetal tissues. Alcohol dehydrogenase of class I was found in most tissues, brain and placenta excluded, but expression levels among tissues differed widely. The distribution pattern of class III transcripts was consistent with those of housekeeping enzymes while, in contrast, class IV transcripts were found only in stomach. Transcripts of multiple length were detected for most classes and were due to different gene products arising through the use of different poly-A signals or transcription from different gene loci. Both class II and class V showed a pattern of liver-enriched expression. However, low mRNA levels were detected also in stomach, pancreas and small intestine for class II, and in fetal kidney and small intestine for class V. Significantly higher levels of class V transcripts were present in fetal liver when compared with levels in adult liver, which suggests that human class V is a predominantly fetal alcohol dehydrogenase.

The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115residue. The Ala-and Asp-t15 mutant proteins were expressed in Escherichia coli and purified by affinity chromatography and ion-exchange HPLC. ITo whom reprint requests should be addressed. 2491The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The tissue distribution of mRNA of alcohol dehydrogenases of classes I, II and III, and sorbitol dehydrogenase, was studied. mRNA from 19 different rat tissues was purified and analyzed by Northern blots, utilizing cDNA probes specific for the four dehydrogenases. Class‐I alcohol‐dehydrogenase mRNA was shown to be of widespread occurrence, detectable in all tissues including brain, but with pronounced differences in amounts. Hybridization revealed the pattern of occurrence of class‐II alcohol‐dehydrogenase mRNA to be unique, with transcripts only in the liver, duodenum, kidney, stomach, spleen and testis. Abundant levels of class‐III alcohol‐dehydrogenase (glutathione‐dependent formaldehyde dehydrogenase) mRNA were present in all tissues analyzed, reflecting the general need for scavenging of formaldehyde in physiological cytoprotection. Sorbitol dehydrogenase mRNA was detected in all tissues except small intestine, in agreement with sorbitol resorbtion by passive diffusion in this tissue. In addition, evidence for a sex‐specific expression, in the liver, of class‐II alcohol dehydrogenase was obtained.

Human class III alcohol dehydrogenase (with both glutathione-dependent formaldehyde dehydrogenase and alcohol dehydrogenase activities) was expressed, and studied by site-directed mutagenesis corresponding to three amino acid residues that are affecting the substrate-binding pocket of class I (with alcohol dehydrogenase activity only). A Thr48Ala exchange results in an enzyme essentially without alcohol dehydrogenase activity but with some glutathione-dependent formaldehyde dehydrogenase activity retained. This indicates that coordination to the enzyme of S-hydroxymethylglutathione is mediated by interactions additional to, or different from, those utilized for primary and secondary alcohols. An Asp57Leu mutation causes considerable loss of the formaldehyde dehydrogenase activity, showing that a negative charge at position 57 is a prerequisite for this class III-type of activity, in the same manner as a positive charge at position 115 has been previously demonstrated to be crucial. Therefore, Asp57 and Arg115 appear to contribute equally to the interactions with S-hydroxymethylglutathione, compatible with defining the class III-type of specificity and possibly explaining the dependence on glutathione. A Tyr93Phe mutant exhibits decreased kcat values for substrates in general and correlates with inhibition of alcohol dehydrogenase activity by 4-methylpyrazole, a potent inhibitor of the class I enzymes. In a double mutant, Asp57Leu/Tyr93Phe, the effects of the two mutations are potentiating one another, yielding a fall in kcat/Km for hydroxymethylglutathione by a factor of 1250, i.e., a still further loss of class III-type activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol dehydrogenases of classes I (the classical liver enzyme) and I11 (formaldehyde dehydrogenase) constitute a pair of moderately related enzymes (63% residue identity between the human forms) that differ fundamentally in many respects. To elucidate the nature of the differences, we have characterized alcohol dehydrogenase from the most primitive vertebrate line (a cyclostome, Atlantic Hagfish), related that to the multiplicity of the human enzyme, and submitted the enzymes to in vitro hybridization for evaluation of subunit interactions. Three findings illustrate important principles of the enzyme system. First, the alcohol dehydrogenase purified from cyclostomes is a class-I11 protein, compatible with the facts that cyclostomes constitute the earliest extant vertebrate line and that class I11 has a distant pre-vertebrate origin. Second, the hagfish enzyme shows multiplicity, with acidic forms in decreasing yield and with amino acid sequences identical between two major isoforms, both aspects constituting properties similar to those of the corresponding human forms. The chemically different subunits are present as homodimers and heterodimers of unmodified and modified subunits, suggesting that the class-I11 multiplicity derives from modification of a type common to lines as divergent as mammals and cyclostomes. Third, the human enzyme can form cross-species hybrid dimers in vitro with the cod and hagfish or Drosophilu class-I11 enzymes (positional identity with the human form of 82, 76 and 70%, respectively). Hence, the results provide experimental evidence for little class-I11 divergence in the segments of subunit interactions. The extent of conservation of residues directly involved in the formation of the subunit interface also reveals a clearly different pattern between classes I and 111. This highlights separation of divergent forms in an enzyme system, with the constant form (class 111) resembling house-keeping enzymes, and exhibiting a correlation between subunit-interacting and substrate-interacting segments.Mammalian alcohol dehydrogenases of apparently six different classes are known [l]. They differ considerably in primary structure (sequence identity, 50-67 %), substrate specificity, tissue distribution and genetic regulation. Knowledge is by far most extensive for the class-I and class-I11 enzymes. The former is the classical liver alcohol dehydrogenase, with considerable ethanol dehydrogenase activity, while the latter constitutes the glutathione-dependent formaldehyde dehydrogenase. Multiple forms within both classes increase the complexity of the enzyme system still further.The class-I enzyme exhibits multiplicity at different levels. One level is derived from gene duplications [2], with resultant subunits explaining the isozymes, long known, for the human enzyme [3]. Another level derives from allelic variability [4], while a third is explained by secondary modifications that have been characterized to only a limited extent in humans and some mammals [3,. The mammalian class-I11 enzymes als...

All four cysteine ligands to the structural zinc atom of human class-I and class-I11 alcohol dehydrogenase have been exchanged by site-directed mutagenesis in order to study the importance of the metal in the mammalian enzymes. The cysteine residues were replaced with Ala and Ser, residues that are not able to ligand zinc. All mutations resulted in inactive, unstable enzymes, in contrast to the non-mutated human alcohol dehydrogenases that are easily isolated. Northern-blot analysis revealed the presence of the expected mRNAs from expression plasmids constructed with the different mutated and non-mutated alcohol dehydrogenases, and Western-blot analysis gave faint signals for the mutated recombinant proteins from crude extracts. This verifies that the plasmid constructs are correct, but that the translated, mutated proteins lacking the zinc-stabilized local fold, are subject to rapid degradation. Hence, the results directly illustrate the importance of the structural zinc atom in mammalian alcohol dehydrogenase and confirm it as a component with 'structural' properties. The results are compatible with those from sensitivities to proteases and from the structures of other proteins within the super-family, indicating that the structural role of the zinc atom may involve conservation of interfaces regulating the enzyme quaternary structure.

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Peptide generation and fast atom bombardment mass spectrometry in combination with conventional chemical analysis was used to identify the blocking group and establish the N-terminal structure of six different proteins at the nanomole level. In this manner, the first terminal structures of three non-mammalian alcohol dehydrogenases were determined, demonstrating the presence of N-terminal acetylation in these piscine, amphibian, and avian enzymes. Similarly, two different yeast glucose-dphosphate dehydrogenases and a minor variant of a human alcohol dehydrogenase were found to be acetylated. The exact end location of C-terminal structures was also established. Together, the analyses permit the definition of terminal regions and blocking groups, thus facilitating the delineation of remaining structures.