Active site liver alcohol dehydrogenase

The Protein Data Bank PDB ID 1A71 Colby T D Bahnson B J Chin J K Klinman J P Goldstein B M Active Site Modifications m a Double Mutant of Liver Alcohol Dehydrogenase Structural Studies of Two Enzyme Ligand Com plexes To be published... 

Schneider, G., Eklund, H., Cedergren-Zeppezauer, E., and Zeppezauer, M. (1983). Crystal structures of the active site in specifically metal-depleted and cobalt-substituted horse liver alcohol dehydrogenase derivatives. Proc. Natl. Acad. Set. U.S.A. 80, 5289-5293. 

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 . 

Figure 16 2 Sketch of the active site of horse liver alcohol dehydrogenase. [Courtesy of C.-I. Branden.]...

These enzymes, which mainly catalyze hydrolytic reactions, have the zinc ions at their active sites. However, Zn ions also appear necessary in some cases for stabilization of the protein structure, e.g. in Cu/Zn SOD, insulin, liver alcohol dehydrogenase and alkaline phosphatases. 

Figure 3-24. A zinc(ii) complex which acts as a functional model for the hydride transfer reaction which occurs at the active site of the enzyme liver alcohol dehydrogenase.

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 ... 

The substrate binding pocket of horse liver alcohol dehydrogenase comprises residues from both subunits (Fig. 26B) [123]. The active site is shown in Fig. 27, with NAD(H) bound, and p-bromobenzyl alcohol bound in a non-productive binding mode. The hydrophobic residues (from both subunits) that line the substrate binding... 

Fig. 26. (A) Schematic diagram of one subunit of horse liver alcohol dehydrogenase. Znl is the active-site zinc. Designed by B. Furugren, from the work of Branden and colleagues [55], (B) Schematic diagram of a section through the horse liver alcohol dehydrogenase dimer. The catalytic zinc atoms are shown, with the inhibitory substrate analogue DMSO and coenzyme molecules indicated. The dimer has two active sites, each composed of parts of both subunits. From the work of Branden and colleagues [123].

Fig. 32. Horse liver alcohol dehydrogenase. The full line shows positions in the apo-enzyme (Fig. 30) and the broken line shows positions in the ternary complex with NADH and dimethyl sulphoxide (Fig. 31). This figure therefore illustrates the movement of active site residues that occurs when the coenzyme and substrate analogue bind. Stereo drawing from the work of Branden and colleagues. 

Tapia and Eklund (1986) carried out a Monte Carlo simulation of the substrate channel of liver alcohol dehydrogenase, based on the X-ray diffraction structure for this enzyme. The addition of substrate and the associated conformation change induce an order—disorder transition for the solvent in the channel. A solvent network, connecting the active-site zinc ion and the protein surface, may provide the basis for a proton relay system. A molecular dynamics simulation of carbonic anhydrase showed two proton relay networks connecting the active-site zinc atom to the surrounding solvent (Vedani et ai, 1989). They remain intact when the substrate, HCOf, is bound. 

Hard electrophiles like Mg(C104)2 are used to activate abiotic systems. In the enzyme liver alcohol dehydrogenase (LAD) a considerably different catalytic apparatus is present a zinc ion coordinated to two cysteines and a histidine serves as a coordinating site for the carbonyl compound/alcoholate, as illustrated in equation (10). This zinc ion has amphoteric properties consistent with the capacity to activate the reaction in both directions without being consumed, in other words to act as a catalyst. Synthetic models of this catalytically active zinc have been shown to possess some catalytic activity in analogy to the enzyme (see Section L3.3.5.1iii). 

Complex (94) (the corresponding complex is insoluble) will induce the same reduction with turnovers of about 100 on the complex. This complex approximates the coordination about zinc in the active site of liver alcohol dehydrogenase. ... 

Dutler, Hans, "Substrate Orientation in the Active Site of Liver Alcohol Dehydrogenase", ibid, pp. 339-350. 

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. 

Opening and closing of active-site region (hexokinase, liver alcohol dehydrogenase, l-arabinose binding protein)... 

In a more specialized case, Horjales and Branden (90) determined a preferred orientation of cyclohexanol in the active site of liver alcohol dehydrogenase for what was believed to be a productive enzyme-substrate complex. By using the positions of the atoms of the bound cyclohexanol ring as a starting point, an extended diamond lattice was constructed to fill the available space in the enzyme site. Each of the lattice points could then be examined to determine the steric possibility of placing an atom at that position. This framework could then be used to design and dock additional structures. 

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... 

Active site modifications in a double mutant of liver alcohol dehydrogenase structural studies of two enzyme-ligand complexes. Biochemistry 37, 9295-9304. 

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]. 

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