<Emphasis Type="Italic">Cupriavidus necator</Emphasis> JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase

Ethanol is a renewable biofuel, and it can be produced from lignocellulosic biomass. The biomass is usually converted to hydrolysates that consist of sugar and sugar derivatives, such as furfural. Yeast ferments sugar to ethanol, but furfural higher than 3 mM is inhibitory. It can take several days for yeast cells to reduce furfural to non-inhibitory furfuryl alcohol before producing ethanol. Bioreduction of furfural to furfuryl alcohol before fermentation may relieve yeast from furfural toxicity. We observed that Cupriavidus necator JMP134, a strict aerobe, rapidly reduced 17 mM furfural to less than 3 mM within 14 min with cell turbidity of 1.0 at 600 nm at 50°C. The rapid reduction consumed ethanol. The “furfural reductase” (FurX) was purified, and it oxidized ethanol to acetaldehyde and reduced furfural to furfuryl alcohol with NAD⁺ as the cofactor. The protein was identified with mass spectrometry fingerprinting to be a hypothetical protein belonging to Zn-dependent alcohol dehydrogenase family. The furX-inactivation mutant of C. necator JMP134 lost the ability to rapidly reduce furfural, and Escherichia coli producing recombinant FurX gained the ability. Thus, an alcohol dehydrogenase enabled bacteria to rapidly reduce furfural with ethanol as the reducing power.     

Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions

The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD⁺ and NADPH/NADP⁺ ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.     

Furfural reduction mechanism of a zinc-dependent alcohol dehydrogenase from Cupriavidus necator JMP134

FurX is a tetrameric Zn-dependent alcohol dehydrogenase (ADH) from Cupriavidus necator JMP134. The enzyme rapidly reduces furfural with NADH as the reducing power. For the first time among characterized ADHs, the high-resolution structures of all reaction steps were obtained in a time-resolved manner, thereby illustrating the complete catalytic events of NADH-dependent reduction of furfural and the dynamic Zn(2+) coordination among Glu66, water, substrate and product. In the fully closed conformation of the NADH complex, the catalytic turnover proved faster than observed for the partially closed conformation due to an effective proton transfer network. The domain motion triggered by NAD(H) association/dissociation appeared to facilitate dynamic interchanges in Zn(2+) coordination with substrate and product molecules, ultimately increasing the enzymatic turnover rate. NAD(+) dissociation appeared to be a slow process, involving multiple steps in concert with a domain opening and reconfiguration of Glu66. This agrees with the report that the cofactor is not dissociated from FurX during ethanol-dependent reduction of furfural, in which ethanol reduces NAD(+) to NADH that is subsequently used for furfural reduction.     

Novel mitochondrial alcohol metabolizing enzymes of <Emphasis Type="Italic">Euglena gracilis</Emphasis>

Ethanol is one of the most efficient carbon sources for Euglena gracilis. Thus, an in-depth investigation of the distribution of ethanol metabolizing enzymes in this organism was conducted. Cellular fractionation indicated localization of the ethanol metabolizing enzymes in both cytosol and mitochondria. Isolated mitochondria were able to generate a transmembrane electrical gradient (Δψ) after the addition of ethanol. However, upon the addition of acetaldehyde no Δψ was formed. Furthermore, acetaldehyde collapsed Δψ generated by ethanol or malate but not by D-lactate. Pyrazole, a specific inhibitor of alcohol dehydrogenase (ADH), abolished the effect of acetaldehyde on Δψ, suggesting that the mitochondrial ADH, by actively consuming NADH to reduce acetaldehyde to ethanol, was able to collapse Δψ. When mitochondria were fractionated, 27% and 60% of ADH and aldehyde dehydrogenase (ALDH) activities were found in the inner membrane fraction. ADH activity showed two kinetic components, suggesting the presence of two isozymes in the membrane fraction, while ALDH kinetics was monotonic. The ADH Km values were 0.64–6.5 mM for ethanol, and 0.16–0.88 mM for NAD⁺, while the ALDH Km values were 1.7–5.3 μM for acetaldehyde and 33–47 μM for NAD⁺. These novel enzymes were also able to use aliphatic substrates of different chain length and could be involved in the metabolism of fatty alcohol and aldehydes released from wax esters stored by this microorganism.     

Effect of alternative NAD<Superscript>+</Superscript>-regenerating pathways on the formation of primary and secondary aroma compounds in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> glycerol-defective mutant

Saccharomyces cerevisiae maintains a redox balance under fermentative growth conditions by re-oxidizing NADH formed during glycolysis through ethanol formation. Excess NADH stimulates the synthesis of mainly glycerol, but also of other compounds. Here, we investigated the production of primary and secondary metabolites in S. cerevisiae strains where the glycerol production pathway was inactivated through deletion of the two glycerol-3-phosphate dehydrogenases genes (GPD1/GPD2) and replaced with alternative NAD⁺-generating pathways. While these modifications decreased fermentative ability compared to the wild-type strain, all improved growth and/or fermentative ability of the gpd1Δgpd2Δ strain in self-generated anaerobic high sugar medium. The partial NAD⁺ regeneration ability of the mutants resulted in significant amounts of alternative products, but at lower yields than glycerol. Compared to the wild-type strain, pyruvate production increased in most genetically manipulated strains, whereas acetate and succinate production decreased in all strains. Malate production was similar in all strains. Isobutanol production increased substantially in all genetically manipulated strains compared to the wild-type strain, whereas only mutant strains expressing the sorbitol producing SOR1 and srlD genes showed increases in isoamyl alcohol and 2-phenyl alcohol. A marked reduction in ethyl acetate concentration was observed in the genetically manipulated strains, while isobutyric acid increased. The synthesis of some primary and secondary metabolites appears more readily influenced by the NAD⁺/NADH availability. The data provide an initial assessment of the impact of redox balance on the production of primary and secondary metabolites which play an essential role in the flavour and aroma character of beverages.     

Cytosolic NADPH balancing in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> cultivated on mixtures of glucose and ethanol

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary ¹³C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD⁺ dependent. Metabolic modeling shows that changing the NAD⁺-aldehyde dehydrogenase to NADP⁺-aldehyde dehydrogenase can increase the penicillin yield on substrate.     

Metabolism of lactose by Clostridium thermolacticum growing in continuous culture

The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l⁻¹) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h⁻¹. Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h⁻¹. The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD⁺, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H₂ removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H₂ scavenging and acetate producing microorganisms.     

An electrochemical aptasensor electrocatalyst for detection of thrombin

This work reports a novel signal amplification strategy based on three-dimensional ordered macroporous C₆₀-poly(3,4-ethylenedioxythiophene)-1-butyl-3-methylimidazolium hexafluorophosphate (3DOM C₆₀-PEDOT-[BMIm][BF₆]) for ultrasensitive detection of thrombin by cascade catalysis of Au-PEDOT@SiO₂ microspheres and alcohol dehydrogenase (ADH). Au-PEDOT@SiO₂ microspheres were constructed not only as nanocarriers to anchor the large amounts of secondary thrombin aptamers but also as nanocatalysts to catalyze the oxidation of ethanol efficiently. Significantly, the electrochemical signal was greatly enhanced based on cascade catalysis: First, ADH catalyzed the oxidation of ethanol to acetaldehyde with the concomitant generation of NADH in the presence of β-nicotinamide adenine dinucleotide hydrate (NAD⁺). Then, gold nanoparticles (AuNPs) as nanocatalysts could effectively catalyze NADH to produce NAD⁺ with the help of PEDOT as redox probe. Under the optimal conditions, the proposed aptasensor exhibits a linear range of 2 × 10⁻¹³ to 2 × 10⁻⁸ M with a low detection limit of 2 × 10⁻¹⁴ M for thrombin detection and shows high sensitivity and good specificity.     

Elimination of glycerol and replacement with alternative products in ethanol fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is a major by-product of ethanol fermentation by Saccharomyces cerevisiae and typically 2–3% of the sugar fermented is converted to glycerol. Replacing the NAD⁺-regenerating glycerol pathway in S. cerevisiae with alternative NADH reoxidation pathways may be useful to produce metabolites of biotechnological relevance. Under fermentative conditions yeast reoxidizes excess NADH through glycerol production which involves NADH-dependent glycerol-3-phosphate dehydrogenases (Gpd1p and Gpd2p). Deletion of these two genes limits fermentative activity under anaerobic conditions due to accumulation of NADH. We investigated the possibility of converting this excess NADH to NAD⁺ by transforming a double mutant (gpd1∆gpd2∆) with alternative oxidoreductase genes that might restore the redox balance and produce either sorbitol or propane-1,2-diol. All of the modifications improved fermentative ability and/or growth of the double mutant strain in a self-generated anaerobic high sugar medium. However, these strain properties were not restored to the level of the parental wild-type strain. The results indicate an apparent partial NAD⁺ regeneration ability and formation of significant amounts of the commodity chemicals like sorbitol or propane-1,2-diol. The ethanol yields were maintained between 46 and 48% of the sugar mixture. Other factors apart from the maintenance of the redox balance appeared to influence the growth and production of the alternative products by the genetically manipulated strains.     

Ethanol exposure modulates hepatic S-adenosylmethionine and S-adenosylhomocysteine levels in the isolated perfused rat liver through changes in the redox state of the NADH/NAD system

Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD⁺ ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high-performance liquid chromatography. The increase in the NADH/NAD⁺ ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD⁺-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD⁺ ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD⁺ ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD⁺ redox couple and the effects of ethanol on methionine metabolism in the liver.     

Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD⁺-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys²⁷⁴→Arg Asn²⁷⁶→Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.     

The Increase in the Number of Accessible SH-Groups in the Enterococcal Membrane Vesicles by ATP and Nicotinamide Adenine Dinucleotides

The number of accessible SH groups was determined in membrane vesicles prepared from Enterococcus hirae grown under anaerobic conditions at alkaline pH (pH 8.0). Addition of ATP or nicotinamide adenine dinucleotides (NAD⁺+NADH) to the vesicles caused a ∼4-fold or ∼1.9-fold increase in the number of SH-groups, respectively. This was inhibited by treatment with N-ethylmaleimide. The increase was significant when ATP and NAD⁺+NADH both were added. The change was lacking in the presence of the F₀F₁-ATPase inhibitors N,N′-diclohexylcarbodiimide or sodium azide. This was also absent in atp mutant with defect in the F₀F₁-ATPase and, in addition, it was less in potassium ion–free medium. These results are correlated with data about K⁺-dependent F₀F₁-ATPase activity, suggesting a relationship between the F₀F₁-ATPase and K⁺ uptake Trk-like system. The latter may be regulated by NAD or NADH mediating conformational changes.     

Photochemical and enzymatic synthesis of methanol from formaldehyde with alcohol dehydrogenase from Saccharomyces cerevisiae and water-soluble zinc porphyrin

We evaluated the photochemical and enzymatic synthesis of methanol from formaldehyde with alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae and NAD⁺ photoreduction by the visible-light sensitization of zinc tetraphenylporphyrin tetrasulfonate (ZnTPPS) in the presence of methylviologen (MV²⁺), diaphorase, and triethanolamine (TEOA). When the sample solution containing ZnTPPS, MV²⁺, NAD⁺, diaphorase, and TEOA in potassium phosphate buffer solution was irradiated, the NADH produced increased with the irradiation time. After irradiation for 180 min, the conversion yield of NAD⁺ to NADH was about 60% under 0.1 mM NAD⁺ condition. The methanol production also depended on the conversion yield of NAD⁺ to NADH. After irradiation for 180 min, 0.38 μM of methanol was produced from formaldehyde (16 μM). The conversion ratio of formaldehyde to methanol was about 2.3%. This result indicates that a system for the photochemical synthesis of methanol from formaldehyde was developed with ADH and the NADH produced by the photosensitization of ZnTPPS in water media.     

Artificial consortium that produces riboflavin regulates distribution of acetoin and 2,3‐butanediol by Paenibacillus polymyxa CJX518

The introduction of an NADH/NAD⁺ regeneration system can regulate the distribution between acetoin and 2,3‐butanediol. NADH regeneration can also enhance butanol production in coculture fermentation. In this work, a novel artificial consortium of Paenibacillus polymyxa CJX518 and recombinant Escherichia coli LS02T that produces riboflavin (VB₂) was used to regulate the NADH/NAD⁺ ratio and, consequently, the distribution of acetoin and 2,3‐butanediol by P. polymyxa. Compared with a pure culture of P. polymyxa, the level of acetoin was increased 76.7% in the P. polymyxa and recombinant E. coli coculture. Meanwhile, the maximum production and yield of acetoin in an artificial consortium with fed‐batch fermentation were 57.2 g/L and 0.4 g/g glucose, respectively. Additionally, the VB₂ production of recombinant E. coli could maintain a relatively low NADH/NAD⁺ ratio by changing NADH dehydrogenase activity. It was also found that 2,3‐butanediol dehydrogenase activity was enhanced and improved acetoin production by the addition of exogenous VB₂ or by being in the artificial consortium that produces VB₂. These results illustrate that the coculture of P. polymyxa and recombinant E. coli has enormous potential to improve acetoin production. It was also a novel strategy to regulate the NADH/NAD⁺ ratio to improve the acetoin production of P. polymyxa.     

Alcohol dehydrogenase polymorphism inDrosophila: Enzyme kinetics of product inhibition

Summary Because natural populations ofDrosophila melanogaster are polymorphic for different allozymes of alcohol dehydrogenase (ADH) and becauseD. melanogaster is more tolerant to the toxic effects of ethanol than its sibling speciesD. simulans, information regarding the sensitivities of the different forms of ADH to the products of ethanol degradation are of ecological importance. ADH-F, ADH-S, ADH-71k ofD. melanogaster and the ADH ofD. simulans were inhibited by NADH, but the inhibition was relieved by NAD⁺. The order of sensitivity of NADH was ADH-F<ADH-71k, ADH-S<ADH-simulans with ADH-F being about four times less sensitive than theD. melanogaster enzymes and 12 times less sensitive than theD. simulans enzyme. Acetaldehyde inhibited the ethanolto-acetaldehyde activity of the ADHs, but at low acetaldehyde concentrations ethanol and NAD⁺ reduced the inhibition. ADH-71k and ADH-F were more subject to the inhibitory action of acetaldehyde than ADH-S and ADH-simulans, with ADH-71k being seven times more sensitive than ADH-S. The pattern of product inhibition of ADH-71k suggests a rapid equilibrium random mechanism for ethanol oxidation. Thus, although the ADH variants only differ by a few amino acids, these differences exert a far larger impact on their intrinsic properties than previously thought. How differences in product inhibition may be of significance in the evolution of the ADHs is discussed.     

Molecular Cloning and Characterization of a Malic Enzyme Gene from the Oleaginous Yeast <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>

The malic enzyme-encoding cDNA (GQ372891) from the oleaginous yeast Lipomyces starkeyi AS 2.1560 was isolated, which has an 1719-bp open reading frame flanked by a 290-bp 5′ untranslated sequence and a 92-bp 3′ untranslated sequence. The proposed gene, LsME1, encoded a protein with 572 amino acid residues. The protein presented 58% sequence identity with the malic enzymes from Yarrowia lipolytica CLIB122 and Aspergillus fumigatus Af293. The LsME1 gene was cloned into the vector pMAL-p4x to express a fusion protein (MBP-LsME1) in Escherichia coli TB1. The fusion protein was purified and then cleaved by Factor Xa to give the recombinant LsME1. This purified enzyme took either NAD⁺ or NADP⁺ as the coenzyme but preferred NAD⁺. The K _m values for malic acid, NAD⁺ and NADP⁺ were 0.85 ± 0.05 mM, 0.34 ± 0.08 mM, and 7.4 ± 0.32 mM, respectively, at pH 7.3.     

The alpha beta epimerization of reduced nicotinamide adenine dinucleotide.

The proton magnetic resonance spectra of the dihydronicotinamide ring of αNADH³ and the nicotinamide ring of αNAD⁺ are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of αNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-αNADH from enzymatically prepared B-deuterio-βNADH. The C4 proton absorption of αNAD⁺ is assigned by oxidation of B-deuterio-αNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterio-αNAD⁺.The epimerization of either αNADH or βNADH yields an equilibrium ratio of approximately 9:1 βNADH to αNADH. The rate of epimerization of αNADH to βNADH at 38 °C in 0.05, pH 7.5, phosphate buffer is 3.1 × 10^?3 min^?1, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M₄ and H₄ lactate dehydrogenase, are shown to oxidize αNADH to αNAD⁺ at rates three to four orders of magnitude slower than for βNADH. By using specifically labeled B-deuterio-αNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of βNADH. The nonenzymatic epimerization of αNADH to βNADH and the enzymatic oxidation αNADH are discussed as a possible source of αNAD⁺in vivo.     

A cold-active and thermostable alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, <Emphasis Type="Italic">Flavobacterium frigidimaris</Emphasis> KUC-1

An NAD⁺-dependent alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 was purified to homogeneity with an overall yield of about 20% and characterized enzymologically. The enzyme has an apparent molecular weight of 160k and consists of four identical subunits with a molecular weight of 40k. The pI value of the enzyme and its optimum pH for the oxidation reaction were determined to be 6.7 and 7.0, respectively. The enzyme contains 2 gram-atoms Zn per subunit. The enzyme exclusively requires NAD⁺ as a coenzyme and shows the pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NAD⁺. F. frigidimaris KUC-1 alcohol dehydrogenase shows as high thermal stability as the enzymes from thermophilic microorganisms. The enzyme is active at 0 to over 85°C and the most active at 70°C. The half-life time and k _cat value at 60°C were calculated to be 50 min and 27,400 min⁻¹, respectively. The enzyme also shows high catalytic efficiency at low temperatures (0–20°C) (k _cat/K _m at 10°C; 12,600 mM⁻¹ min⁻¹) similar to other cold-active enzymes from psychrophiles. The alcohol dehydrogenase gene is composed of 1,035 bp and codes 344 amino acid residues with an estimated molecular weight of 36,823. The sequence identities were found with the amino acid sequences of alcohol dehydrogenases from Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%) and Geobacillus stearothermophilus LLD-R (56%). This is the first example of a cold-active and thermostable alcohol dehydrogenase.     

2-Mercaptoethanol as a substrate for liver alcohol dehydrogenase.

An activity was identified in a phosphate buffer extract of calf liver acetone powder which utilized 2-mercaptoethanol and NAD⁺ as substrates and formed NADH as one product. The activity responsible for catalyzing this reaction is associated with calf liver alcohol dehydrogenase based on copurification, similarity in pH optima, and similarity in response to chelating agents and other inactivating agents. Crystalline horse liver alcohol dehydrogenase also catalyzes the formation of NADH from NAD⁺ using 2-mercaptoethanol as the substrate. Although the K_m for mercaptoethanol is much lower than that for ethanol, 30 μm as compared to 0.625 mm, the maximum velocity with mercaptoethanol as the substrate is only 7% of that when ethanol is the substrate. Because of this difference in maximum velocity, 2-mercaptoethanol is an apparent competitive inhibitor with respect to ethanol with crystalline horse liver alcohol dehydrogenase, consistent with ethanol and 2-mercaptoethanol binding at the same site. The apparent K_i for 2-mercaptoethanol is 14 μm. 2-Butanethiol is a competitive inhibitor with respect to both 2-mercaptoethanol and ethanol with horse and beef liver alcohol dehydrogenases.