Alcohol Dehydrogenase

I.U.B.: 1.1.1.1
Alcohol: NAD+ oxidoreductase

Alcohol dehydrogenase catalyzes the reaction:

The common reaction in yeast cells is reduction of acetaldehyde to ethanol. In vitro the enzyme is generally assayed and used in a more alkaline pH region, a condition which favors a shift of equilibrium towards the oxidation of ethanol.

Characteristics of Alcohol Dehydrogenase from Yeast:

Molecular weight: 141,000 comprised of four subunits of 35,000 each (Büchner and Sund 1969).

Composition: A metalloenzyme containing four tightly bound zinc atoms per molecule (Vallee and Hoch 1955). Per subunit, there are two distinct active site sulfhydryl groups which can be distinguished on the basis of differential reactivity with iodoacetate and butyl isocyanate (Twu, Chin, and Wold 1973). A histidine residue is considered to have an essential role (Dickenson and Dickinson 1975).

Optimum pH: For the oxidation of ethanol, pH 8.6-9.0 (the enzyme becomes increasingly unstable at higher pH). For the reduction of acetaldehyde a pH nearer to 7.0 is considered optimum. This reaction is kinetically complex with pH being only one factor determining optimum conditions.

Extinction coefficient: = 12.6.

Isoelectric point: 5.4 (Sund and Theorell 1963).

Activators: Sulfyhydryl activating reagents, mercaptoethanol, dithiothreitol, cysteine, etc., and heavy metal chelating reagents.

Specificity: Yeast ADH which has a more narrow specificity than that of liver enzyme, accepts ethanol, is somewhat active on the straight chain primary alcohols, and acts to a very limited extent on certain secondary and branched chain alcohols (Dickinson and Dalziel 1967). NADP does not serve as coenzyme.

Inhibitors: Heavy metals and -SH reagents. See Sund and Theorell (1963) for a more comprehensive list; also Anderson and Reynolds (1966), Anderson et al. (1966), Atkinson et al. (1967), Fiddick and Heath (1967), Rashed and Rabin (1968).

Stabilizers: Dilute solution of the enzyme may be stabilized by serum albumin, gelatin, and/or glutathione or cysteine. At pH values below 6.0 and above 8.5 the enzyme is increasingly unstable. More concentrated solutions of the enzyme in high purity water, near neutrality, are stable several days at 5 °C.

Stability: Lyophilized preparations, stored refrigerated, are stable 6-12 months. Crystalline suspensions in ammonium sulphate are stable 6 months at 2 - 8 °C.

Characteristics of Alcohol Dehydrogenase from Horse Liver:

Molecular weight: 80,000 (Drum et al. 1967; Blomquist et al. 1967).

Composition: A dimer enzyme comprised of two distinctive subunit types, E (for "ethanol-active"), and S (for "steroid-active") which differ in relative substrate specificities (Jörnvall, 1970, Andersson et al. 1974). There are two firmly bound zinc atoms and one active center per subunit. The primary sequence of the E type subunit containing 374 residues has been reported by Jörnvall (1970). In commercial preparations of horse liver alcohol dehydrogenase (HLADH) the EE isoenzyme is predominant. In addition to the occurrence of secondary modified forms, Pietrusyko (1974) has reported animal to animal variation of HLADH. See also Jörnvall et al. (1990).

Sytkowski and Vallee (1975) report on the replacement of zinc atoms by cobalt. See also Veillon and Sytkowski (1975), Reynolds and McKinley-McKee (1974 and 1975), Coleman et al. (1972).

Active site investigations include: Canella and Sodini (1975), Chen and Engel (1975), Dworschack et al. (1975), Hadorn et al. (1975), Ward and Cull (1974), Coleman and Weiner (1973), Hansch et al. (1972).

HLADH activity and kinetics have been described by Blackwell and Hardman (1975), Cronholm et al. (1975), Favilla and Cavatorta (1975), Fries et al. (1975), McFarland and Chu (1975), Shore et al. (1974 and 1975), Tatemoto (1975).

Extinction coefficient: = 4.55 (Cannon and McKay 1969).

Isoelectric point: 6.8 (Dalziel 1958).

Activators: A study has shown that enzyme activity can be greatly enhanced by imidoesters or cyanate (Plapp 1970). Glutathione and EDTA have activating or protective effects towards heavy metal ion inhibitors.

Specificity: Liver ADH possesses a broad specificity. See review by Sund and Theorell (1963), Dickinson and Dalziel (1967), and Reynier et al. (1969). See also Hardman et al. (1974), Bessman and McCabe (1972), and Hinson and Neal (1972).

Inhibitors: The enzyme is inhibited by heavy metals, sulfhydryl reagents and zinc chelating agents (Brand et al. 1967). See also Gunnarsson et al. (1974), Lange et al. (1974), Li and Magnes (1972), Cheng and Lek (1992), Miwa et al. (1987), and Skurksky et al. (1992).

Stability: Both the lyophilized and crystalline suspension preparations are stable for 3-6 months when stored at -20°C.