Hydrophobic anion activation of human liver chi chi alcohol dehydrogenase

Class III alcohol dehydrogenase (chi chi-ADH) from human liver binds both ethanol and acetaldehyde so poorly that their Km values cannot be determined, even at ethanol concentrations up to 3 M. However, long-chain carboxylates, e.g., pentanoate, octanoate, deoxycholate, and other anions, substantially enhance the binding of ethanol and other substrates and hence the activity of class III ADH up to 30-fold. Thus, in the presence of 1 mM octanoate, ethanol displays Michaelis-Menten kinetics. The degree of activation depends on the size both of the substrate and of the activator; generally, longer, negatively charged activators result in greater activation. At pH 10, the activator binds to the E-NAD+ form of the enzyme to potentiate substrate binding. Pentanoate activates methylcrotyl alcohol oxidation and methylcrotyl aldehyde reduction 14- and 30-fold, respectively. Such enhancements of both oxidation and reduction are specific for class III ADH; neither class I nor class II shows this effect. The implications as to the nature of the physiological substrate(s) of class III ADH are discussed in light of the recent finding that this ADH and glutathione-dependent formaldehyde dehydrogenase are identical. A new rapid purification procedure for chi chi-ADH is presented.     

Physical and enzymatic properties of a class III isozyme of human liver alcohol dehydrogenase: chi-ADH

chi-Alcohol dehydrogenase (chi-ADH), a class III isozyme characterized by its anodic electrophoretic mobility and lack of inhibition by 4-methylpyrazole, has been isolated from human liver and purified to homogeneity in a reducing medium. chi-ADH resembles other human liver ADH isozymes of classes I and II with respect to its molecular weight, dimeric structure, stoichiometry of zinc and NADH binding, and pH optima for the oxidation of alcohols. This homodimer exhibits subtle differences in its absorption spectrum and amino acid composition relative to those of other human isozymes but differs markedly from their specificity toward alcohols and aldehydes. chi-ADH oxidizes ethanol very poorly. The reaction is bimolecular, and an apparent Km cannot be discerned up to 2.3 M ethanol. The enzyme is inactive toward methanol, ethylene glycol, digitoxigenin, digoxigenin, and gitoxigenin , but alcohols with carbon chain lengths greater than four are oxidized rapidly with Km values decreasing with increasing carbon chain length. Taken jointly, the composition, structure, and enzymatic properties of the ADH isozymes purified and studied so far strongly imply that their metabolic roles, yet to be discovered, will give a new perspective to ethanol metabolism and pathology.     

Evidence for the identity of glutathione-dependent formaldehyde dehydrogenase and class III alcohol dehydrogenase

Formaldehyde dehydrogenase (EC 1.2.1.1) is a widely occurring enzyme which catalyzes the oxidation of S-hydroxymethylglutathione, formed from formaldehyde and glutathione, into S-formyglutathione in the presence of NAD. We determined the amino acid sequences for 5 tryptic peptides (containing altogether 57 amino acids) from electrophoretically homogeneous rat liver formaldehyde dehydrogenase and found that they all were exactly homologous to the sequence of rat liver class III alcohol dehydrogenase (ADH-2). Formaldehyde dehydrogenase was found to be able at high pH values to catalyze the NAD-dependent oxidation of long-chain aliphatic alcohols like n-octanol and 12-hydroxydodecanoate but ethanol was used only at very high substrate concentrations and pyrazole was not inhibitory. The amino acid sequence homology and identical structural and kinetic properties indicate that formaldehyde dehydrogenase and the mammalian class III alcohol dehydrogenases are identical enzymes.     

Organ specific alcohol metabolism: placental chi-ADH

Human placenta contains a single detectable isozyme of alcohol dehydrogenase that has been isolated and characterized. It migrates toward the anode on starch gel electrophoresis and can be stained with pentanol but not ethanol as substrate. Its kinetic and molecular characteristics are identical with those of the recently discovered chi-ADH (Class III) isozyme from human liver. Placental ADH is present in the cytosol of this organ in small amounts, 6 mg/kg fresh tissue. It oxidizes ethanol very slowly--even at ethanol concentrations that would reflect intoxication when found in serum. Thus, placental alcohol dehydrogenase cannot play a significant role in the ethanol metabolism of pregnant women.     

Purification, characterization, and partial sequence of the glutathione-dependent formaldehyde dehydrogenase from Escherichia coli: a class III alcohol dehydrogenase.

The glutathione-dependent formaldehyde dehydrogenase from Escherichia coli has been purified to homogeneity and characterized. It is a 83,000-kDa homodimer containing 4 g-atom of zinc per dimer with a specific activity of 60 units/mg toward S-(hydroxymethyl)glutathione and NAD+ as substrates. Its isoelectric point, 4.4, is consistent with both its amino acid composition and chromatographic behavior on DEAE HPLC. The N-terminus is unblocked, and 47 residues from the N-terminus were sequenced. A computer search of the Swiss-Prot protein sequence data bank shows that the N-terminal sequence, [sequence; see text], is homologous with the mammalian class III alcohol dehydrogenases with 27 identities when compared to the human enzyme. Like the human, rat, and rabbit enzymes, it has high formaldehyde dehydrogenase activity in the presence of glutathione and catalyzes the oxidation of normal alcohols (ethanol, octanol, 12-hydroxydodecanoate) in a reaction that is not GSH-dependent. In addition, hemithiolacetals other than those formed from GSH, including omega-thiol fatty acids, also are substrates. The wide distribution and high degree of similarity of this enzyme to the plant and animal alcohol dehydrogenases suggest that the E. coli enzyme is closely related to the ancestor of the plant and animal dimeric zinc alcohol dehydrogenases.     

Amphibian alcohol dehydrogenase, the major frog liver enzyme. Relationships to other forms and assessment of an early gene duplication separating vertebrate class I and class III alcohol dehydrogenases

Submammalian alcohol dehydrogenase structures can be used to evaluate the origins and functions of the different types of the mammalian enzyme. Two avian forms were recently reported, and we now define the major amphibian alcohol dehydrogenase. The enzyme from the liver of the Green frog Rana perezi was purified, carboxymethylated, and submitted to amino acid sequence determination by peptide analysis of six different digests. The protein has a 375-residue subunit and is a class I alcohol dehydrogenase, bridging the gap toward the original separation of the classes that are observable in the human alcohol dehydrogenase system. In relation to the human class I enzyme, the amphibian protein has residue identities exactly halfway (68%) between those for the corresponding avian enzyme (74%) and the human class III enzyme (62%), suggesting an origin of the alcohol dehydrogenase classes very early in or close to the evolution of the vertebrate line. This conclusion suggests that these enzyme classes are more universal among animals than previously realized and constitutes the first real assessment of the origin of the duplications leading to the alcohol dehydrogenase classes. Functionally, the amphibian enzyme exhibits properties typical for class I but has an unusually low Km for ethanol (0.09 mM) and Ki for pyrazole (0.15 microM) at pH 10.0. This correlates with a strictly hydrophobic substrate pocket and one amino acid difference toward the human class I enzyme at the inner part of the pocket. Coenzyme binding is highly similar, while subunit-interacting residues, as in other alcohol dehydrogenases, exhibit several differences.(ABSTRACT TRUNCATED AT 250 WORDS)     

A human liver alcohol dehydrogenase enzyme-linked immunosorbent assay method specific for class I, II, and III isozymes

A sensitive and convenient method for the quantitative measurement of human alcohol dehydrogenase (ADH) isozymes based on enzyme-linked immunosorbent assay has been devised. The procedure was optimized with respect to antigen coating density, antiserum dilution, and incubation times with rabbit antisera raised against beta 1 beta 1-ADH to achieve a limit of sensitivity of 1 ng/ml for this isozyme when purified. Using the optimal conditions established, quantitative measurement of alpha beta 1, alpha gamma 1, beta 1 gamma 1, pi, and chi-ADH were obtained with antisera raised in rabbits toward these individual isozymes. The incorporation into the procedure of thimerosal (ethyl(4-mercaptobenzoato-S)mercury) or other sulfhydryl specific reagents improved the soluble phase antiserum avidity for all ADH isozymes, thereby increasing the sensitivity. Thimerosal is an absolute requirement for chi-ADH antigen-antibody binding. The polyclonal rabbit antisera elicited by the individual isozymes of the three classes of ADH exhibit a high degree of isozyme class specificity. Cross-reactivity of the antibodies with the beta 1 beta 1, alpha gamma 1, alpha gamma 2, alpha beta 1, beta 1 gamma 1, beta 1 gamma 2, pi and chi isozymes were evaluated. Antisera against the class I isozymes beta 1 beta 1 and beta 1 gamma 1 cross-react with all class I isozymes and with pi-ADH. Antibodies against pi and chi-ADH are selective and specific only for their respective antigens. Neither one cross-reacts with any class I isozyme. Conformational effects resulting from subunit interactions likely account for differences in cross-immunoreactivity between the closely homologous class I isozymes.     

Maize glutathione-dependent formaldehyde dehydrogenase cDNA: a novel plant gene of detoxification

We have previously shown that intact plants and cultured plant cells can metabolize and detoxify formaldehyde through the action of a glutathione-dependent formaldehyde dehydrogenase (FDH), followed by C-1 metabolism of the initial metabolite (formic acid). The cloning and heterologous expression of a cDNA for the glutathione-dependent formaldehyde dehydrogenase from Zea mays L. is now described. The functional expression of the maize cDNA in Escherichia coli proved that the cloned enzyme catalyses the NAD⁺- and glutathione (GSH)-dependent oxidation of formaldehyde. The deduced amino acid sequence of 41 kDa was on average 65% identical with class III alcohol dehydrogenases from animals and less than 60% identical with conventional plant alcohol dehydrogenases (ADH) utilizing ethanol. Genomic analysis suggested the existence of a single gene for this cDNA. Phylogenetic analysis supports the convergent evolution of ethanol-consuming ADHs in animals and plants from formaldehyde-detoxifying ancestors. The high structural conservation of present-day glutathione-dependent FDH in microorganisms, plants and animals is consistent with a universal importance of these detoxifying enzymes.     

Sequence of the gene for a NAD(P)-dependent formaldehyde dehydrogenase (class III alcohol dehydrogenase) from a marine methanotroph Methylobacter marinus A45

Abstract A fragment of Methylobacter marinus A45 DNA has been cloned and sequenced, and an open reading frame has been identified that could code for a 46-kDa polypeptide. Comparison of the deduced amino acid sequence of the polypeptide against the protein data bank has revealed strong similarity with a number of alcohol dehydrogenases, with highest similarity towards class III alcohol dehydrogenases, which recently have been shown to be identical to glutathione-dependent formaldehyde dehydrogenases. We were unable to measure appreciable levels of NAD(P)-dependent formaldehyde dehydrogenases or alcohol dehydrogenase activities using aldehydes or primary or secondary alcohols in cell-free extracts from batch cultures of M. marinus A45. However, formaldehyde dehydrogenases activity was detected on zymograms. Our data suggest that, although NAD(P)-linked formaldehyde dehydrogenase or alcohol dehydrogenase activities are undetectable in cell-free extracts of most methylotrophs employing the ribulose monophosphate pathway for formaldehyde assimilation and dissimilation, the gene encoding formaldehyde dehydrogenase is present in M. marinus A45 and may be present in more of these organisms as well.     

Alcohol dehydrogenase from Methylobacterium organophilum.

The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 x 10(-5) M for methanol and 8.2 x 10(-5) M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum.     

Purification and characterization of a new alcohol dehydrogenase from human stomach

Starch gel electrophoresis of homogenates from human stomach mucosa resolves three alcohol dehydrogenase (ADH) forms: the anodic chi-ADH (class III), the cathodic gamma-ADH (class I), and a new form of slow cathodic mobility that has not been previously characterized. In this work, we describe the purification in three chromatographic steps and the physical and kinetic characterization of this new human alcohol dehydrogenase, which we have named sigma-ADH. The enzyme exhibits the general physicochemical features (Mr, zinc content, subunit Mr, cofactor preference) of all mammalian alcohol dehydrogenases. The kinetic studies show a high Km value (41 mM) and a high kcat value (280 min-1) for ethanol at pH 7.5. The Km decreases as the alcohol increases its chain length. The aldehydes are better substrates than the corresponding alcohols, with m-nitrobenzaldehyde being the best substrate examined. sigma-ADH is strongly inhibited by 4-methylpyrazole, but with a Ki (10 microM) still higher than that for a class I isoenzyme. These properties suggest that sigma-ADH is a class II isoenzyme, different from pi-ADH and similar to that previously described by us in rat stomach. At the high ethanol concentrations in stomach after drinking, sigma-ADH is probably the ADH form with the largest contribution to human gastric ethanol metabolism.     

NAD- and co-substrate (GSH or factor)-dependent formaldehyde dehydrogenases from methylotrophic microorganisms act as a class III alcohol dehydrogenase

Abstract The methylotrophic yeasts, Hansenula polymorpha and Candida boidinii , and the methylotrophic Gram-negative bacteria, Paracoccus denitrificans and Thiobacillus versutus (but not Methylophaga marina ), contain NAD/GSH-dependent formaldehyde dehydrogenase when grown on C₁-compounds. The enzymes appeared to be similar to each other and to the mammalian counterparts with respect to substrate specificity, including the ability to act as an alcohol dehydrogenase class III. The Gram-positive bacteria, Amycolatopsis methanolica and Rhodococcus erythropolis , possess NAD/Factor-dependent formaldehyde dehydrogenase when grown on C₁-compounds or on C₁-unit-containing substrates, respectively. These enzymes also exhibit alcohol dehydrogenase class III activity. Thus, like the mammalian ones, methylotrophic formaldehyde dehydrogenases show dual substrate specificity, suggesting that this is an inherent property of the enzyme.     

Cobalt(II)-substituted class III alcohol and sorbitol dehydrogenases from human liver

The catalytic zinc atoms in class III (chi) alcohol dehydrogenase (ADH) and sorbitol dehydrogenase (SDH) from human liver have been specifically removed and replaced by cobalt(II) with a new ultrafiltration technique. The electronic absorption spectrum of class III cobalt ADH (epsiolon 638 = 870 M-1 cm-1) is nearly identical with those of active site substituted horse EE and human class I (beta 1 beta 1) cobalt ADH. Thus, the coordination environment of the catalytic metal is strictly conserved in these enzymes. However, significant differences are noted when the spectra of class III ADH-coenzyme complexes are compared to the corresponding spectra of the horse enzyme. The spectrum of class III ADH.NADH is split into three bands, centered at 680, 638, and 562 nm. The class III ADH.NAD+ species resembles the alkaline form of the corresponding horse enzyme complex but without exhibiting the pH dependence of the latter. These spectral changes underscore the role of the coenzymes in differentially fine tuning the catalytic metal for its particular function in each ADH. The noncatalytic zinc of class III ADH exchanges with cobalt at pH 7.0. While 9 residues out of 15 in the loop surrounding the noncatalytic zinc of class III ADH differ from those of the class I ADH, the electronic absorption spectra of cobalt in the noncatalytic metal site of class III ADH establish that the coordination environment of this site is conserved as well. The spectrum of cobalt SDH differs significantly from those of cobalt ADHs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase

The class III human liver alcohol dehydrogenase, identical to glutathione-dependent formaldehyde dehydrogenase, separates electrophoretically into a major anodic form (₁) of known structure, and at least one minor, also anodic but a slightly faster migrating form (₂). The primary structure of the minor form isolated by ion-exchange chromatography has now been determined. Results reveal an amino acid sequence identical to that of the major form, suggesting that the two derive from the same translation product, with the minor form modified chemically in a manner not detectable by sequence analysis. This pattern resembles that for the classical alcohol dehydrogenase (class I). Hence, the ₁/₂ multiplicity does not add further primary forms to the complex alcohol dehydrogenase system but shows the presence of modified forms also in class III.     

Class III human liver alcohol dehydrogenase: a novel structural type equidistantly related to the class I and class II enzymes

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)     

Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase

Formaldehyde, a major industrial chemical, is classified as a carcinogen because of its high reactivity with DNA. It is inactivated by oxidative metabolism to formate in humans by glutathione-dependent formaldehyde dehydrogenase. This NAD(+)-dependent enzyme belongs to the family of zinc-dependent alcohol dehydrogenases with 40 kDa subunits and is also called ADH3 or chi-ADH. The first step in the reaction involves the nonenzymatic formation of the S-(hydroxymethyl)glutathione adduct from formaldehyde and glutathione. When formaldehyde concentrations exceed that of glutathione, nonoxidizable adducts can be formed in vitro. The S-(hydroxymethyl)glutathione adduct will be predominant in vivo, since circulating glutathione concentrations are reported to be 50 times that of formaldehyde in humans. Initial velocity, product inhibition, dead-end inhibition, and equilibrium binding studies indicate that the catalytic mechanism for oxidation of S-(hydroxymethyl)glutathione and 12-hydroxydodecanoic acid (12-HDDA) with NAD(+) is random bi-bi. Formation of an E.NADH.12-HDDA abortive complex was evident from equilibrium binding studies, but no substrate inhibition was seen with 12-HDDA. 12-Oxododecanoic acid (12-ODDA) exhibited substrate inhibition, which is consistent with a preferred pathway for substrate addition in the reductive reaction and formation of an abortive E.NAD(+).12-ODDA complex. The random mechanism is consistent with the published three-dimensional structure of the formaldehyde dehydrogenase.NAD(+) complex, which exhibits a unique semi-open coenzyme-catalytic domain conformation where substrates can bind or dissociate in any order.     

The metabolic role of human ADH3 functioning as ethanol dehydrogenase

Human class III alcohol dehydrogenase (ADH3), also known as glutathione-dependent formaldehyde dehydrogenase, exhibited non-hyperbolic kinetics with ethanol at a near physiological pH 7.5. The S(0.5) and k(cat) were determined to be 3.4+/-0.3 M and 33+/-3 min(-1), and the Hill coefficient (h) 2.21+/-0.09, indicating positive cooperativity. Strikingly, the S(0.5) for ethanol was found to be 5.4 x 10(6)-fold higher than the K(m) for S-(hydroxymethyl)glutathione, a classic substrate for the enzyme, whereas the k(cat) for the former was 41% lower than that for the latter. Isotope effects on enzyme activity suggest that hydride transfer may be rate-limiting in the oxidation of ethanol. Kinetic simulations using the experimentally determined Hill constant suggest that gastric ADH3 may highly effectively contribute to the first-pass metabolism at 0.5-3 M ethanol, an attainable range in the gastric lumen during alcohol consumption. The positive cooperativity mainly accounts for this metabolic role of ADH3.     

Oxidation of thiodiglycol (2,2'-thiobis-ethanol) by alcohol dehydrogenase: comparison of human isoenzymes

Sulfur mustard is a chemical warfare agent that causes blistering of the skin and damages the eyes and airway after environmental exposure. We have previously reported that thiodiglycol (TDG, 2,2'-bis-thiodiethanol), the hydrolysis product of sulfur mustard, is oxidized by alcohol dehydrogenase (ADH) purified from horse liver or present in mouse liver and human skin cytosol. Humans express four functional classes of ADH composed of several different isozymes, which vary in their tissue distribution, some occurring in skin. To help us evaluate the potential contribution of the various human isozymes toward toxicity in skin and in other tissues, we have compared the catalytic activity of purified human class I alphaalpha-, beta1beta1-, beta2beta2-, and gamma1gamma1-ADH, class II pi-ADH, class III chi-ADH, and class IV sigma-ADH with respect to TDG oxidation and their relative sensitivities to inhibition by pyrazole. Specific activities toward TDG were 123, 79, 347, 647, and 12 nmol/min/mg for the class I alphaalpha-, beta1,beta1-, beta2beta2-, and gamma1gamma1-ADH and class II pi-ADH, respectively. TDG was not a substrate for class III chi-ADH. The specific activity of class IV sigma-ADH was estimated at about 1630 nmol/min/mg. 1 mM pyrazole, a potent inhibitor of class I ADH, inhibited the class I alphaalpha, beta1beta1, beta2beta2, and gamma1gamma1 ADH and class IV sigma-ADH by 83, 100, 56, 90, and 73%, respectively. The class I alphaalpha- and beta1beta1-ADH oxidized TDG with kcat/Km value of 7-8 mM(-1) min(-1), beta2beta2-ADH with a value 19 mM(-1) min(-1) and class I gamma1gamma1-ADH with a value of 176 mM(-1) min(-1). The kcat/Km value for class IV sigma-ADH was estimated at 4 mM(-1) min(-1). The activities of class IV sigma-ADH and class I gamma1gamma1-ADH are of significant interest because of their prevalence in eyes, lungs, stomach, and skin, all target organs of sulfur mustard toxicity.     

Rabbit liver alcohol dehydrogenase: isolation and characterization of class I isozymes

Livers of rabbits contain three classes of alcohol dehydrogenase (ADH) isozymes which are highly analogous to the human classes. Class I ADHs migrate toward cathode on starch gel and are very sensitive to 4-methylpyrazole (4-MePz) inhibition. Class II ADH migrates slowly toward anode and is less sensitive to 4-MePz. Class III ADH migrates rapidly toward anode and is insensitive to 4-MePz. There are one class II, one class III and at least three class I ADH isozymes present in the rabbit liver. The three class I isozymes purified to homogeneity are all dimers with subunit molecular weight of 41700. Two are heterodimers composed of A-, C-chains and B-, C-chains, respectively. The third one is a homodimer, contains only the C-chain. These results indicate that among all the mammals examined, rabbit ADH bears the greatest resemblance to the human enzyme.     

Alcohol Dehydrogenase from Methylobacterium organophilum

The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10⁻⁵ M for methanol and 8.2 × 10⁻⁵ M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum.