N-Alkane oxidation enzymes of a pseudomonad.

A nicotinamide adenine dinucleotide (NAD)-dependent n-alkane dehydrogenase and an NAD phosphate (reduced form)-dependent alkane hydroxylase have been purified from cell-free extracts of Pseudomonas sp. strain 196Aa grown anaerobically on n-alkane. The n-alkane dehydrogenase (fraction R-3), obtained as a single peak from Bio-Gel P-60, showed an overall 135-fold purification and was demonstrated by infrared spectroscopy and gas chromatography to convert n-decane to 1-decene. The alkene hydroxylase activity in the S-3 fraction, purified 167 times from diethylaminoethyl-cellulose, was shown by the same methodology to convert decene to decanol. Commercial ferredoxin has been shown to increase the alkane dehydrogenase activity. An NAD-, flavine adenine dinucleotide-, and iron-dependent alcohol dehydrogenase was demonstrated in the R-3 fraction. A mechanism for the anaerobic conversion of n-alkane to fatty acid has been proposed.     

Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

Interaction of nad with rape alcohol dehydrogenase

Rape alcohol dehydrogenase is competitively inhibited with respect to NAD by nicotinamide, as well as by compounds containing adenine (adenine, adenosine, AMP, ADP, ATP). Adenine and adenosine are bound more firmly to the enzyme than nicotinamide. The two types of compound, as component parts of the NAD coenzyme, are bound to different sites on the enzyme. Adenine and adenosine compete for the adenine nucleotide bonding site, but they do not compete for the o-phenanthroline bonding site. Nicotinamide competes with o-phenanthroline for the binding site at which the metal is apparently present.     

Nucleotide pyrophosphatase from yeast. The presence of bound zinc.

Nucleotide pyrophosphatase from yeast was inhibited by thiols, o-phenanthroline, 8-hydroxyquinoline, EDTA, and 8-hydroxyquinoline-5-sulfonic acid. The inhibition by chelating agents was time and concentration dependent. Inhibition by EDTA was decreased by complexing the EDTA with metal ions before addition to the enzyme. The effectiveness of the metal ions in preventing inhibition by EDTA paralleled the stability constants of the EDTA-metal complexes. Partial recovery of EDTA-inhibited enzyme activity was achieved with Zn²⁺, Co²⁺, Fe²⁺, and Mn²⁺. Analyses for zinc in the purified enzyme by atomic absorption spectroscopy and by titration with 8-hydroxyquinoline-5-sulfonic acid revealed the presence of approximately 1 g atom/mol of enzyme (M_r 65,000). The data indicate that yeast nucleotide pyrophosphatase is a metalloenzyme in which the zinc plays some role in activity.     

Studies of yeast alcohol dehydrogenase with 3-aminopyridine mononucleotide

Summary 3-Aminopyridine mononucleotide, a nicotinamide mononucleotide analog, was prepared by enzymatic cleavage of 3-aminopyridine adenine dinucleotide by a snake venom phosphodiesterase and isolated by means of ion exchange chromatography. The spectrophotometric and fluorometric properties of this analog were studied. Several anions were shown to quench the fluorescence intensity of this analog. pH was shown to have a pronounced effect on the fluorescence intensity. 3-Aminopyridine mononucleotide was shown to be a coenzyme-competitive inhibitor of yeast alcohol dehydrogenase. The 3-aminopyridine mononucleotide was diazotized with the use of nitrous acid. A time dependent irreversible inactivation of yeast alcohol dehydrogenase resulted from incubation with the diazotized 3-aminopyridine mononucleotide at pH 7.0. Incubation of the enzyme with NAD prior to the addition of the diazotized 3-aminopyridine mononucleotide protected the enzyme against inactivation.Recently, 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP), NAD and NADP analogs respectively, were synthesized by either chemical or enzymatic processes. The chemical, spectrophotometric properties of these dinucleotides have also been reported. It was demonstrated that these nucleotides serve as coenzyme-competitive inhibitors of dehydrogenases but did not function as coenzymes for oxidation-reduction reactions catalyzed by these enzymes. The pyridine amino group of AAD was diazotized and the diazotized derivative was shown to inactive yeast alcohol dehydrogenase irreversibly. Isolation of modified cysteine residue from the modified yeast alcohol dehydrogenase resulting from inactivation by diazotized AAD has been reported. Thus, diazotized AAD proved to be a site specific label for the coenzyme binding site of yeast alcohol dehydrogenase. It was of interest to prepared and determine the properties of a NMN analog, 3-aminopyridine mononucleotide (APMN). The preparation of APMN was accomplished by enzymatic cleavage of AAD with snake venom phosphodiesterase according to a method previously reported. This report deals with the preparation, properties and studies of APMN with yeast alcohol dehydrogenase.This work was supported in part by Research Grant GR-IX from Old Dominion University Research Foundation.     

Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high-performance immobilized metal ion affinity chromatography

High-performance immobilized metal ion affinity chromatography was utilized to evaluate the adsorption properties of 67 synthetic, biologically active, peptides ranging in size from 5 to 42 residues. The metal ions, Cu(II), Ni(II) and Zn(II), were immobilized by iminodiacetic acid (IDA) coupled to TSK gel 5PW (10 microns). Two types of gradient elution (imidazole and pH) were used to evaluate peptide retention by the metal ions. A decreasing pH gradient and an increasing imidazole gradient eluted the peptides in similar order. IDA-Cu(II) and IDA-Zn(II) showed very similar selectivities for the peptides analyzed; however, IDA-Zn(II) displayed a weaker affinity for the peptides. IDA-Ni(II) showed a slightly different pattern of selectivity. Peptide adsorption effects contributed by the metal-free gel matrix were found to be relatively minor. The concentration and type of salt included in the mobile phase could affect the relative affinities of the peptides for the immobilized metal ions. Retention coefficients were assigned to individual amino acid residues by multiple linear regression analysis. Histidine showed the largest positive correlation with retention, followed by aromatic amino acid residues. Modified N-terminal residues resulted in negative contributions to retention. Analyses of peptide amino acid composition alone allowed prediction of peptide retention behavior on immobilized metal ion affinity columns.     

The binding of 1,10-phenanthroline to specifically active-site cobalt(II)-substituted horse-liver alcohol dehydrogenase. A probe for the open-enzyme conformation

We have studied the binding of 1,10-phenanthroline to specifically active-site cobalt(II)-substituted horse-liver alcohol dehydrogenase [Co(II)-LADH]. The dissociation constant is a factor of 6500 smaller than in the native enzyme. Spectral evidence is given which shows that 1,10-phenanthroline does not remove the catalytic Co(II) ion and that binding of 1,10-phenanthroline renders the catalytic metal ion pentacoordinate. The maximum limiting rate constant for the association of 1,10-phenanthroline to Co(II)-LADH is about 60 s-1. This is about a third of the value (169 s-1) determined for native horse-liver alcohol dehydrogenase, Zn(II)LADH [Frolich et al. (1978) Arch. Biochem. Biophys. 189, 471-480]. For cadmium(II)-substituted horse-liver alcohol dehydrogenase, [Cd(II)LADH] the maximum limiting rate constant for association of 1,10-phenanthroline increased to 590 s-1. These findings demonstrate that the rate-limiting step is strongly dependent on the chemical nature of the catalytic metal ion and its immediate environment. 1,10-Phenanthroline is shown to bind to the Co(II)-LADH.NAD+ complex in the open conformation. The maximum limiting rate constant remains unchanged in the presence of NAD+. The data have been used to derive a kinetic scheme for the formation of ternary complexes including NAD+ that involves a slow intermediary step.     

Mutation of surface-exposed histidine residues of recombinant human granulocyte-colony stimulating factor (Cys17Ser) impacts on interaction with chelated metal ions and refolding in aqueous two-phase systems

Site directed mutagenesis of Cys17-->Ser17 form of recombinant human granulocyte colony stimulating factor (rhG-CSF C17S) for sequential replacing of surface His(43) and His(52) with alanine was used to identify residues critical for the protein interaction with metal ions, in particular Ni(2+) chelated by dye Light Resistant Yellow 2 KT (LR Yellow 2KT)-polyethyleneglycol (PEG), and refolding after partitioning of inclusion bodies in aqueous two-phase systems. Strong binding of rhG-CSF (C17S) to PEG-LR Yellow 2KT-Cu(II) complex allowed for the adoption of affinity chromatography on Sepharose-LR Yellow 2KT-Cu(II) that appeared to be essential for the rapid isolation of mutated forms of rhG-CSF. Efficiency of that purification stage is exemplified by isolation of rhG-CSF (C17S, H43A) and rhG-CSF (C17S, H43A, H52A) mutants in correctly folded and highly purified state. Affinity partitioning of rhG-CSF histidine mutants was studied in aqueous two-phase systems containing Cu(II), Ni(II) and Hg(II) chelated by LR Yellow 2KT-PEG at pH 7.0 and Cu(II)-at pH 5.0. It was determined, that affinity of rhG-CSF mutants for metal ions decreased in the order of C17S>C17S, H43A>C17S, H43A, H52A for Cu(II), and C17S=C17S, H43A>C17S, H43A, H52A for Ni(II) ions, while affinity of all rhG-CSF mutants for Hg(II) ions was of the same order of magnitude. Influence of His(43) and His(52) mutation on protein refolding was studied by partitioning of the respective inclusion body extract in aqueous two-phase systems containing Ni(II) and Hg(II) ions. Data on rhG-CSF histidine mutant partitioning and refolding indicated, that His(52) mutation is crucial for the strength of protein interaction with chelated Ni(II) ions and refolding efficiency.     

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.     

Metabolism of acetoin in mammalian liver slices and extracts. Interconversion with butane-2,3-diol and biacetyl

1. [(14)C]Acetoin was enzymically synthesized from [(14)C]pyruvate with a pyruvate decarboxylase preparation. Its optical activity was [alpha](20) (d)-78 degrees . 2. Large amounts (1000-fold higher than physiological concentrations) of acetoin were incubated with rat liver mince. Acetoin disappeared but very little (14)CO(2) was evolved. A compound accumulated, which was purified and identified as butane-2,3-diol. Chromatography on borate-impregnated paper indicated the presence of both the erythro and threo forms. 3. Liver extracts capable of interconverting biacetyl, acetoin and butane-2,3-diol were obtained. These interconversions were catalysed by two different enzymes: acetoin dehydrogenase (EC 1.1.1.5) and butane-2,3-diol dehydrogenase (EC 1.1.1.4), previously identified in bacteria. Both required NAD(+) or NADP(+) as cofactors and were different from alcohol dehydrogenase. The equilibrium in both cases favoured the more reduced compound. 4. The activity of butane-2,3-diol dehydrogenase was decreased by dialysis against EDTA: the addition of Co(2+), Cu(2+), Zn(2+) and other bivalent metal ions restored activity. 5. Biacetyl reductase was resolved into multiple forms by CM-Sephadex chromatography and electrophoresis.     

Dissociation of outer-sphere water is rate-limiting for the binding of ligands in the active site of horse liver alcohol dehydrogenase

The association of imidazole and auramine O to native horse-liver alcohol dehydrogenase [Zn(II)LADH] and active-site specifically cobalt(II)-substituted horse-liver alcohol dehydrogenase [Co(II)LADH], respectively, has been investigated. In all cases [except imidazole binding to Zn(II)LADH in the presence of auramine O] the association rates approached an upper limit (kmax). The kmax values were compared for the metal ligands imidazole (monodentate), 1,10-phenanthroline and 2,2'-bipyridine (bidentate; see also the preceding paper), and for auramine O which does not coordinate to the catalytic metal ion. Independent of the large differences in their structure and metal-bonding capability, all these compounds exhibit common, maximum, limiting rate constants of about 60 s-1 and 200 s-1 for Co(II)LADH and Zn(II)LADH, respectively. These results demonstrate that kmax is strongly dependent on the catalytic metal ion but not on the ligand. The absence of spectral changes in the d-d transitions of the catalytic Co(II) ion upon auramine O binding to Co(II)LADH indicates that the rate-limiting step is not accompanied by a major conformational change. Finally, it is concluded that reactions in the inner coordination sphere of the catalytic metal ion (i.e. the metal-bound water molecule) are not responsible for the step characterized by kmax. We propose the rate-limiting step to consist of the dissociation of one or several water molecules from the second coordination sphere of the catalytic metal ion in the active site of LADH in its open conformation.     

Hexuronic acid dehydrogenase of Agrobacterium tumefaciens

Growth of Agrobacterium tumefaciens on d-glucuronic acid (GlcUA) or d-galacturonic acid (GalUA) induces formation of hexuronic acid dehydrogenase [d-aldohexuronic acid: nicotinamide adenine dinucleotide (NAD) oxidoreductase]. The dehydrogenase, which irreversibly converts GlcUA or GalUA to the corresponding hexaric acid with the concomitant reduction of NAD, but not of nicotinamide adenine dinucleotide phosphate was purified 60-fold by MnCl(2) treatment, (NH(4))(2)SO(4) fractionation, chromatography on diethylaminoethyl Sephadex and negative adsorption with Ca(3)(PO(4))(2) gel. The pH optimum is 8.0. Other uronic acids, aldohexoses, aldopentoses, and polyols, are not substrates. Reduced nicotinamide adenine dinucleotide is an inhibitor strictly competitive with NAD. Kinetic data indicate that the dehydrogenase induced by growth on GlcUA may not be identical with that induced by growth on GalUA.     

X-ray absorption edge spectroscopy of Co(II)-binding sites of copper- and zinc-containing proteins

X-ray absorption near-edge spectroscopy (XANES) of Co(II) in three derivatives of superoxide dismutase, namely [Cu(II)-Co(II)], [Cu(I)-Co(II)] and [...-Co(II)], suggests a tetrahedral coordination of the metal for all compounds. Significant differences, detected in the spectrum of the [Cu(II)-Co(II)] derivative as compared to the other species, indicate that a conformational change and/or a different charge of the imidazole bridging the two metal sites in superoxide dismutase occur in coincidence with the change of copper valence. The XANES spectra of the cobalt derivatives of alcohol dehydrogenase, carbonic anhydrase and stellacyanin show features that can be accounted for by an increasing degree of covalency in the metal first sphere of coordination, in the following order: alcohol dehydrogenase greater than stellacyanin greater than superoxide dismutase greater than or equal to carbonic anhydrase.     

Studies of the pH dependence of the formation of binary and ternary complexes with liver alcohol dehydrogenase

The association of the coenzyme NAD+ to liver alcohol dehydrogenase (LADH) is known to be pH dependent, with the binding being linked to the shift in the pK of some group on the protein from a value of 9-10, in the free enzyme, to 7.5-8 in the LADH-NAD+ binary complex. We have further characterized the nature of this linkage between NAD+ binding and proton dissociation by studying the pH dependence (pH range 6-10) of the proton release, delta n, and enthalpy change, delta Ho(app), for formation of both binary (LADH-NAD+) and ternary (LADH-NAD+-I, where I is pyrazole or trifluoroethanol) complexes. The pH dependence of both delta n and delta Ho(app) is found to be consistent with linkage to a single acid dissociating group, whose pK is perturbed from 9.5 to 8.0 upon NAD+ binding and is further perturbed to approximately 6.0 upon ternary complex formation. The apparent enthalpy change for NAD+ binding is endothermic between pH 7 and pH 10, with a maximum at pH 8.5-9.0. The pH dependence of the delta Ho(app) for both binary and ternary complex formation is consistent with a heat of protonation of -7.5 kcal/mol for the coupled acid dissociating group. The intrinsic enthalpy changes for NAD+ binding and NAD+ plus pyrazole binding to LADH are determined to be approximately 0 and -11.0 kcal/mol, respectively. Enthalpy change data are also presented for the binding of the NAD+ analogues adenosine 5'-diphosphoribose and 3-acetylpyridine adenine dinucleotide.     

Parallelism between ethanol and imidazole interactions with horse liver alcohol dehydrogenase

The rate effects of imidazole on the EE isoenzyme of horse liver alcohol dehydrogenase have been analysed in terms of the elucidated kinetic mechanism of the enzyme. These imidazole effects on both directions of the reaction within nonexcess as well as excess ranges of substrate concentrations pointed to the competition between imidazole and ethanol for binding to the same three enzyme species in the kinetic mechanism, namely the free enzyme, the enzyme-NAD+ complex, and the enzyme-NADH complex. Moreover, both imidazole and ethanol brought about an enhancement in the rate of dissociation of NAD+ from its binding site on the enzyme.     

Alcohol dehydrogenase from the fruitfly Drosophila melanogaster. Inhibition studies of the alleloenzymes AdhS and AdhUF

Different metal binding inhibitors of horse liver alcohol dehydrogenase, similarly affect the Drosophila melanogaster AdhS and AdhUF alleloenzymes. However, binding is generally weaker and the experiments show that the alleloenzymes although not zinc metalloenzymes, behave to the metal binding reagents very much as if they were. The metal-directed, affinity-labelling, imidazole derivative BrImPpOH reversibly inhibits, but does not inactivate the alleolenzymes. This confirms there is no active site metal atom with cysteine as a metal ligand, as found in zinc alcohol dehydrogenases. Pyrazole is a strong ethanol-competitive inhibitor of AdhS and AdhUF alleloenzymes. Formation of the ternary enzyme-NAD-pyrazole complex gives an absorption increase between 295-330 nm. This enables an active site titration to be performed and the determination of epsilon (305 nm) of 15.8 . 10(3) M-1 . cm-1. Inhibition experiments with imidazole confirm that with secondary alcohols such as propan-2-ol, a Theorell-Chance mechanism predominates, but with ethanol and primary alcohols, interconversion of the ternary complexes is rate limiting. Salicylate is a coenzyme competitive inhibitor and KEI suggests that the coenzyme adenosine binding region is similar is Drosophila and horse liver alcohol dehydrogenase. Drosophila alcohol dehydrogenase is found not to form a ternary complex with NADH and isobutyramide. In this and other properties it is like carboxymethyl liver alcohol dehydrogenase. Both Drosophila and carboxymethyl alcohol dehydrogenase bind coenzyme in a similar manner to native horse liver alcohol dehydrogenase, but substrate binding differs between each. Inhibition by Cibacrone blue, indicates that amino acid 192 which is lysine in AdhS and threonine in AdhUF, is located in the coenzyme-binding region. Proteolytic activity present in preparations of alcohol dehydrogenase from D. melanogaster, is considered due to a metalloprotease, for which BrImPpOH is a potent inactivator.     

Purification and properties of beta-hydroxybutyrate dehydrogenase from Mycobacterium phlei ATCC354.

beta-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) was purified 145-fold from Mycobacterium phlei ATCC354 by ammonium sulphate fractionation and DEAE-cellulose chromatography. The pH optima for oxidation and reduction reactions were 8.4 and 6.8 respectively. The purified enzyme was specific for NAD, NADH, acetoacetate and D(-)-beta-hydroxybutyrate. Km values for DL-beta-hydroxybutyrate and NAD were 7.4 mM and 0.66 mM respectively. The enzyme was inactivated by mercurial thiol inhibitors and by heat, but could be protected by NADH, Ca2+ and partially by Mn2+. The enzyme did not require metal ions and was insensitive to EDTA, glutathione, dithiothreitol, beta-mercaptoethanol and cysteine.     

Electrochemical evaluation of lactate dehydrogenase immobilized in polyacrylamide gels

Lactate dehydrogenase (EC 1.1.1.27) has been immobilized in polyacrylamide gels over a platinum grid matrix. The immobilized enzyme is used to oxidize L-lactate in the presence of nicotinamide adenine dinucleotide (NAD+) and ferricyanide. The NADH produced is then chemically oxidized back to NAD+ by ferricyanide. The coupled reduction of ferricyanide ions to ferrocyanide ions results in a measurable electrochemical potential. This measurable zero-current potential is found to be Nernstian in nature and directly proportional to the logarithm values of L-lactate concentration over the range of 2 X 10(-5) to 5 X 10(-2)M. The results indicate that immobilized lactate dehydrogenase can be incorporated into a system to detect L-lactate acid in aqueous solutions.     

Synthesis of coenzymically active soluble and insoluble macromolecularized NAD+ derivatives.

Alkylation at N-1 of the NAD+ adenine ring with 3,4-epoxybutanoic acid, followed by chemical reduction to the alkali-stable NADH form and alkaline Dimroth rearrangement, gave the NADH derivative alkylated at the exocyclic adenine amino group. Enzymic reoxidation of the latter derivative gave nicotinamide-6-(2-hydroxy-3-carboxypropylamino)purine dinucleotide, a functionalized NAD+ analogue carrying an omega-carboxyalkyl side-chain at the exocyclic adenine amino group. Carbodiimide coupling of the latter derivative to high-molecular-weight water-soluble (polyethyleneimine, polylysine) and insoluble (aminohexyl-Sepharose) polymers gave the corresponding macromolecularized NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine and polylysine analogues showed a substantial degree of efficiency relative to free NAD+ with rabbit muscle lactate dehydrogenase (60 and 25% respectively) but a lower one with yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase (2-7%). The polyethyleneimine derivative entrapped in cellulose triacetate fibres together with the lactate dehydrogenase was operationally stable during repetitive use.     

Reaction of 4-trans-(N,N-dimethylamino)cinnamaldehyde with the liver alcohol dehydrogenase-oxidized nicotinamide adenine dinucleotide complex

Evidence that horse liver alcohol dehydrogenase forms a ternary complex with 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) and oxidized nicotinamide adenine dinucleotide (NAD+) is presented. Formation of the complex is characterized by a 97-nm red shift of the free chromophore to 495 nm (epsilon 495 approximately 6.0 X 10(4) M-1 cm-1). This shift is larger than the 66-nm red shift of the E(NADH,-DACA) complex (lambda max = 464 nm) previously reported by Dunn and Hutchinson [Dunn, M.F., & Hutchison, J.S. (1973) Biochemistry 12, 4882-4892]. The large red shift of the E(NAD+,DACA) complex is due to the combined effects of coordination of the carbonyl oxygen of DACA to the active-site zinc ion and to the close proximity of the positively charged nicotinamide ring of NAD+. The stability of this complex is pH dependent and depends on a single apparent ionization with pKa = 7.6 +/- 0.3. The pH-independent dissociation constant for binding of DACA to E(NAD+) is 23 +/- 6 microM. The stoichiometry of DACA binding to the E(NAD+) complex is shown to be one per active site (two per enzyme molecule). Liver alcohol dehydrogenase is also shown to catalyze the NAD+-mediated oxidation of DACA to the corresponding carboxylic acid with a very slow turnover rate. The possibility that the observed E(NAD+,DACA) complex is an intermediate in the enzyme-catalyzed oxidation of DACA is discussed.