Liver alcohol dehydrogenase metal complex

Another zinc-utilizing enzyme is carbonate/dehydratase C (Kannan et al., 1972). Here, the zinc is firmly bound by three histidyl side chains and a water molecule or a hydroxyl ion (Fig. 27). The coordination is that of a distorted tetrahedron. Metals such as Cu(II), Co(Il), and Mn(ll) bind at the same site as zinc. Hg(II) also binds near, but not precisely at, this site (Kannan et al., 1972). Horse liver alcohol dehydrogenase (Schneider et al., 1983) contains two zinc sites, one catalytic and one noncatalytic. X-Ray studies showed that the catalytic Zn(II), bound tetrahedrally to two cysteines, one histidine, and water (or hydroxyl), can be replaced by Co(II) and that the tetrahedral geometry is maintained. This is also true with Ni(Il). Insulin also binds zinc (Adams etai, 1969 Bordas etal., 1983) and forms rhombohedral 2Zn insulin crystals. The coordination of the zinc consists of three symmetry-related histidines (from BIO) and three symmetry-related water molecules. These give an octahedral complex... 

Certain transition metal complexes exhibit activating properties and act with turnover on the metal center analogously to the catalytically active zinc ion in the active center of liver alcohol dehydrogenase. Various chiral europium shift reagents, for example Eu(hfc)3, induce reduction of (9b) by 1,4-dihydroni-cotinamides. Turnovers of about 100 are obtained on the metal complexes and methyl mandelate is formed with enantiomeric excesses of 25-44%. ... 

Figure 1-8. Typical co-ordination complexes of transition metal ions in proteins. 1 M may be Fe2+, as in rubredoxin, or Zn2 as in aspartate transcarbamylase and alcohol dehydrogenase, 2 carboxypeptidase A, 3 carbonic anhydrase, 4 liver alcohol dehydrogenase, 5 azurin, 6 heme group, L is His and L either His or Met in cytochromes, 7 deoxy-heme group in hemoglobin and myoglobin, 8 oxyform of 7, 9 superoxide dismutase.

Fig. 6. Sketch of the horse liver alcohol dehydrogenase-adenosine-diphosphoribose (ADPR) binary complex in the region of the active site. ADPR lies in an 20 A-deep cavity which extends to the metal ion as indicated). The second cavity ( 20 A deep also) is believed to be the substrate binding site (47)...

Evidence from fluorescence, absorption, and c.d. spectra suggests that the conformations of various tetracycline antibiotics in complexes with Ca + are different from those in the magnesium complexes. The optical properties of the catalytically active cobalt(n) form of liver alcohol dehydrogenase, which normally requires zinc for activity, indicate an unusual binding environment for the metal (c/. the implications of the absorption and c.d. spectra of the cobalt proteases, which have recently been discussed ). 

The Job method has been used to show that horse-liver alcohol dehydrogenase contains zinc atoms bound in two different environments, and it appears that Zn + plays a functional role in the interaction of DNA with DNA polymerases from E. coli and sea urchins. The nature of the two metal-binding sites in transferrin has been investigated, using tervalent lanthanide ions as fluorescent probes the two sites are different and the distance between them is 43 A or greater. The kinetics of the reaction between Fe -nta and transferrin have been measured by the stopped-flow method and a reaction sequence has been proposed. The same technique, with C1 n.m.r. detection, has been used to study the extraction of the metal from the Hg -bovine serum albumin complex by various ligands. 

Complex kinetics of reactivation were observed for liver alcohol dehydrogenase in the presence of Zn. No association was detectable in the absence of Zn. However, excess of metal led to the formation of incorrectly refolded species (see Section 11.5) (Jaenicke, 1979). 

The NAD+-dependent alcohol dehydrogenase from horse liver contains one catalytically essential zinc ion at each of its two active sites. An essential feature of the enzymic catalysis appears to involve direct coordination of the enzyme-bound zinc by the carbonyl and hydroxyl groups of the aldehyde and alcohol substrates. Polarization of the carbonyl group by the metal ion should assist nucleophilic attack by hydride ion. A number of studies have confirmed this view. Zinc(II) catalyzes the reduction of l,10-phenanthroline-2-carbaldehyde by lV-propyl-l,4-dihy-dronicotinamide in acetonitrile,526 and provides an interesting model reaction for alcohol dehydrogenase (Scheme 45). The model reaction proceeds by direct hydrogen transfer and is absolutely dependent on the presence of zinc(II). The zinc(II) ion also catalyzes the reduction of 2- and 4-pyridinecarbaldehyde by Et4N BH4-.526 The zinc complex of the 2-aldehyde is reduced at least 7 x 105 times faster than the free aldehyde, whereas the zinc complex of the 4-aldehyde is reduced only 102 times faster than the free aldehyde. A direct interaction of zinc(II) with the carbonyl function is clearly required for marked catalytic effects to be observed. 

Electrospray ionization has allowed the observation of a great number of non-covalent complexes protein-protein, protein-metal ion, protein-drug and protein-nucleic acid. About one-third of the proteins exist as multimeric forms. Mass spectrometry allows the study of their quaternary structure. This has been done for alcohol dehydrogenase (ADH) from horse liver and from yeast. The ESI spectra are displayed in Figure 8.22. The horse liver ADH is observed to be dimeric whereas that of yeast is tetrameric [131]. 

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