Liver alcohol dehydrogenase, LADH

For a liver alcohol dehydrogenase (LADH) model an NS2O coordination sphere is required. The chelating aldehydes are ideal for the formation of this donor set when combined with bis(pentafluoro-thiophenolato)zinc. Structural data on the complexes with one equivalent of 6-methylpyridine-2-carbaldehyde, 6-methoxypyridine-2-carbaldehyde, 2-(dimethylamino)benzal-dehyde) demonstrate that the coordination sphere for LADH has been reproduced to a close approximation and the corresponding alcohol complexes have also been characterized.354 Other thiophenols have been used to form such complexes but have not been structurally characterized.304... 

Probably the most extensively studied enzymes are those from alcohol dehydrogenase family. One enzyme from this series which has been thoroughly examined both experimentally and theoretically is liver alcohol dehydrogenase (LADH). It catalyzes the reversible conversion of an alcohol to an aldehyde by transferring hydride from substrate to the cofactor (NAD+) ... 

Alcohol dehydrogenases (ADH EC 1.1.1.1), for which several X-ray structures are available ", catalyze the biological oxidation of primary and secondary alcohols via the formal transfer of a hydride anion to the oxidized form of nicotinamide adenine dinucleotide (NAD ), coupled with the release of a proton. Liver alcohol dehydrogenase (LADH) consists of two similar subunits, each of which contains two zinc sites, but only one site within each subunit is catalytically active. The catalytic zinc is coordinated in a distorted tetrahedral manner to a histidine residue, two cysteine residues and a water molecule. The remaining zinc is coordinated tetrahedrally to four cysteine residues and plays only a structural role . 

The enzyme consists of three main isozymes formed by the dimeric combination of two different protein chains. The two types of protein chains have been labelled E (for ethanol active) and S (for steroid active). About 90% of liver alcohol dehydrogenase (LADH)) is EE and the remaining 10% consists of ES and SS. SS is also ethanol active, although lower than ES and EE, and vice versa. Polymeric forms of the isozymes are also known. 

Studies on the various zinc-activated dehydrogenases continue apace. The reduction of tra s-4-iViV-dimethylaminocinnamaldehyde (A) by liver alcohol dehydrogenase (LADH) is reported to involve the zinc at the active site of the enzyme acting as a Lewis acid and co-ordinating the substrate via the aldehyde oxygen.235 The kinetics of the reaction show that (A) 4- LADH -f NADH form a stable intermediate at pH 9, the overall reaction sequence being ... 

Experimental data on primary and secondary kinetic isotope effects in the hydride-transfer step in liver alcohol dehydrogenase, LADH, were analyzed using canonical variational transition theory (CVT) for overbarrier dynamics and the optimized multidimentional path (OMT) for the nuclear tunneling (Alhambra et al., 2000 and references therein). This work demonstrates somewhat better agreement of theoretical values of primary and secondary Schaad- Swein exponents calculated by combining CVT/OMT methods with the experimental values instead of CVT and classical transition states (TST). 

The efforts to synthesize cobalt species containing two thiolate ligands was stimulated by the fact that catalytic zinc in liver alcohol dehydrogenase (LADH) is coordinated by two cysteines, and cobalt-... 

Similarly, enzyme activity has been correlated to solvent polarity. Oxidation of cinnamyl alcohol by horse liver alcohol dehydrogenase (LADH) (pH 7.5) was observed in anhydrous hexane, methylene chloride and acetonitrile (Guinn et al., 1991). The oxidation rates were observed to increase as the dielectric constant decreased (Table 5). Electron paramagnetic spectroscopy (EPR) and two active site directed spin labels were used to examine the effect of solvent dielectric on structural stability. As the dielectric constant of the solvent decreased, the spectra broadened, indicative of an increase in rigidity or stability. 

Synthesis and structural characterization of synthetic analogs of liver alcohol dehydrogenase (LADH) continued to be one of the most investigated fields of research. A number of ligands suitable for modeling LADH have been reported and their hydroxo zinc complexes also described.133-135 [Zn(TmMes)(HOMe)]+, for example, exhibits a coordination environment that resembles aspects of that in LADH.136 The ethanol complex [Zn(Tm Bu)(HOEt)]+ has been also isolated.137 [NS2] donor ligands, that feature thioether donors, also provide coordination environments that mimic the active site of LADH.138,139... 

Cui, Q., Elstner, M., Karplus, M. (2002) A theoretical analysis of the proton and hydride transfer in liver alcohol dehydrogenase (LADH),... 

Horse liver alcohol dehydrogenase (LADH) catalyzes the reactions of aldehydes and their corresponding alcohols with the coenzymes NADH and NAD+. Activation of substrate complexes via polarization of substrate C=0 bond has been observed in LADH by vibrational spectroscopy. Two enzyme complexes have been studied by difference Raman measurements, the E/NADH DABA complex [17, 18] and the E/NADH CXF complex [19]. DABA is a poor substrate while CXF is a substrate analog. X-ray crystallography has shown that the polarization of the substrate C=0 bond is mainly due to a coordination to the active site Zn++ ion [20, 21]. For example, polarization of the C=0 bond of DABA in the LADH complex was found to be substantial, half way between a single and double bond as compared to DABA in solution [18]. 

Horse Liver Alcohol Dehydrogenase (LADH) Catalysis... 

HORSE LIVER ALCOHOL DEHYDROGENASE (LADH) CATALYSIS... 

These general concepts can be exemplified by liver alcohol dehydrogenases (LADH), dimeric zinc enzymes of 80 kDa that catalyze the following class of reactions using the NADH/NAD system as coenzyme (or, really, as cosubstrate) ... 

Horse liver alcohol dehydrogenase, LADH, provides a somewhat different example of an enzyme in which the intrinsic metal atoms appear... 

Measurements of absorption as a function of time can provide a wide range of useful information. Brzovic and Dunn [8] describe instrumentation for measuring the time dependence of absorption spectra after rapid mixing of reactants. Several rapid-scanning stopped-flow instruments are commercially available reactions that take place in a millisecond or longer can be studied. Enzyme-catalyzed reactions with natural chromophores, such as NADH, are discussed, and the substitution of a colored metal center [Co(II)] for a colorless one [Zn(II)] are also described. Detailed mechanistic conclusions for horse liver alcohol dehydrogenase (LADH) are given. 

The work with liver alcohol dehydrogenase (LADH) is particularly illustrative of the information that can be obtained from a suitable chromophoric probe. Dunn and Hutchison discovered that the LADH-catalyzed reduction of 4-tran5-(N,N-dimethylamino)cinnamaldehyde (DACA) occurs via a transient intermediate wherein the carbonyl oxygen of DACA forms an inner sphere coordination complex with the active site zinc ion. The function of the bonding interaction is to activate the aldehyde carbonyl... 

Fig. 2. Diagram depicting the active site of horse liver alcohol dehydrogenase (LADH) with 4-/ranj-(Af,Af-dimethylamino)cinnamaldehyde coordinated to the active site zinc ion. (From Dunn and Hutchison with permission.) Copyright 1973 American Chemical Society.

Liver alcohol dehydrogenase (LADH) is a dimer with two tryptophan residues in each identical subunit and a total molecular weight of 80 hDa. One of the residues is exposed to the solvent (tip- IS), and one residue is buried (tip-314). This buried residue can be selectively excited on the red edge of the absorption spectrum at 300 nm. ... 

Tolf and colleagues prepared 4-alkynylpyrazoles and their saturated analogs and assayed them for in vitro inhibition of horse liver alcohol dehydrogenase (LADH). It was shown that the saturated substituents displayed increased inhibitory activity (84BAP265). 

Horse liver alcohol dehydrogenase (LADH) catalyzes the reversible... 

Fig. 8.12. Cl longitudinal relaxation rate as a function of the molar ratio of reduced coenzyme (NADH) to horse liver alcohol dehydrogenase (LADH). The solutions contained 0.13 mM LADH and 0.5 M KCl. The pH was 8.4 and the temperature 6°C. (From a study by Bull, Einarsson, Lindman and Zeppezauer [136])...

The mechanism of liver alcohol dehydrogenase (LADH) has been extensively studied. For a recent overview the reader is referred to Ref [93]. Reaction field effects on the transition structure of model hydride transfer systems have been calculated at ab initio 4-3IG basis set level [93, 94]. The active site of enzymes are usually assumed to be designed to receive molecules in the transition state for the reaction they catalyze. This special sort of surrounding medium effects has been computationally documented recently [95]. From the reaction geodesic passing through the transition state for hybride transfer in the pyridium cation/methanolate model system, only the TS-structure could be fitted into the LADH active site. The normal mode analysis carried out on the TS showed an excellent agreement with isotopic substitution experiments [95]. Reaction field calculations on this model systems have also been performed. For an overview of biomolecular interactions the reader is referred to Ref [96]. 

In a previous paper from our laboratory (1), it was pointed out that hydrogen peroxide can act as a substrate for equine liver alcohol dehydrogenase (LADH) (EC 1.1.1.1), giving rise to a catalytic process that has been extensively studied (2-4). 

Horse liver alcohol dehydrogenase (LADH EC 1.1.1.1) is today one of the most thoroughly investigated enz3nnes. Since its crystallization in 1948 (1) many authors have studied its properties in great details the Swedish school, first with Theorell and then with Br anden and their co-workers, has extensively reviewed our knowledges on this and related dehydrogenases (2,3). The active form of LADH is known to be dimeric and its three-dimensional structure has been solved at 2.4 A resolution (4). 

An interesting pair of consecutive reactions involve the absorption of ethyl alcohol by the body, which is a first-order process, and the consequent oxidation of alcohol to acetaldehyde by liver alcohol dehydrogenase (LADH), which is a zeroth order process. The differential changes in the three states of ethanol can therefore be described as... 

---