Liver alcohol dehydrogenase, function

The dimensions of cavities in enzymes differ considerably, depending on their physiological function. In many cases the clefts are occupied by clusters of organized water molecules. Such clusters can be seen in certain X-ray structures of enzymes (e.g., the structure of carboxypeptidase A determined by Lipscomb). If the clefts are deep, as in horse liver alcohol dehydrogenase, a channel for removal of water is present (Branden). 

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.

Fig. 35. Diagrammatic representation of functionally equivalent groups around the substrate in lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and horse liver alcohol dehydrogenase. From the work of Rossmann and colleagues [164],...

Scharschmidt M, Fisher MA, Cleland, WW. Variation of transition-state structure as a function of the nucleotide in reactions catalyzed by dehydrogenases. 1. Liver alcohol dehydrogenase with benzyl alcohol and yeast aldehyde dehydrogenase with benzalde-hyde. Biochemistry 1984 23 5471-5478. 

Zinc deficiency in humans is difficult to diagnose because a suitable functional test, or a test for zinc reservoirs, has been lacking, Milne et ai. (1987) documented that a variety of zinc-requiring enzymes of the red blood cell seem to resist the effects of dietary zinc deficiency, but demonstrated that liver alcohol dehydrogenase may be sensitive to the deficiency. The activity of this enzyme was assessed by a functional test — the ethanol tolerance test. The ethanol tolerance test would be expected to be influenced by factors unrelated to zinc status, such as the state of Induchon of the ethanol oxidizing system, and hence may not be the functional test of choice for assessing zinc status. 

Fig. 11. Schematic representation of the interactions between the substrate, coenzyme, and the active site residues in horse liver alcohol dehydrogenase. Not shown are the interactions between Arg-47 and the pyrophosphate backbone, and Asp-49, which forms a salt bridge with His-57, another ligand of the zinc atom. Because of the close proximity to residues having obvious catalytically important functions, alterations in the interactions between the coenzyme and Ser-48 and His-51 that are anticipated from the binding of acyclo-NAD could readily cause the observed changes in substrate specificity. Based on Ref. 38.

There have been extensive discussions in the literature regarding maximization of the catalytic efficiency of enzymes and the value of their internal equilibrium constants is the equilibrium constant between substrates and products of the enzyme when all are bound productively) (98-102). For example, the value of A int is near unity for both liver alcohol dehydrogenase (78) and lactate dehydrogenase (103) when measured with their natural substrates. The ability of these same enzymes to function with alternative substrates with widely differing external equilibrium constants raises important questions regarding the relationships of the internal thermodynamics of such reactions. 

Previously known substrates have been reviewed earlier (f) as well as the widespread occurrence of alcohol dehydrogenase in nature. Alcohols, including ethanol, produced in the intestinal tracts mainly by bacterial actions, are found in the portal vein. One physiological function of liver alcohol dehydrogenase may be to metabolize these products 8). 

The discovery of the steroid-active isozymes of horse liver alcohol dehydrogenase (Sections II,A,l,a and II,A,l,d) also established that 3-keto-and 3 3-hydroxysteroids are substrates, but the functional significance of this is not clear. Since this activity also seems to be present in the rat enzyme (9) and in all isozymes of the human enzyme (W), it may be more important than previously realized when the horse ethanol-active isozyme was the most studied form. It may also be noted that mammalian livers contain many different specific steroid dehydrogenases (11). Structural studies have established that mammalian alcohol dehydrogenases have a distant evolutionary link to both the yeast (ff) and bacterial enzymes (IS). 

Fig. 2. Reasonable structural explanation for the stereospecilicity of hydrogen transfer catalyzed by horse liver alcohol dehydrogenase. The binary complexes were obtained by building ethanol into the X-ray structure of the holoenzyme, on the basis of the assumption that the ethanolic oxygen is directly coordinated to the active site zinc ion. (A) When the pro-(R) proton of ethanol is directed at C-4 of the nicotinamide ring, the methyl function is favorably positioned in the active site (B) when the pro-(S) proton is directed at C-4, the methyl function interacts stcrically with Phe-93. (From Ref. 30), with permission.]...

Today, for the purpose of illustrating these points, we wish to review selected segments of work performed in our laboratory on two multichain enzymes—alkaline phosphatase from . coli and equine liver alcohol dehydrogenase—in which intrinsic metal atoms appear to play both functional and structural roles. Thereafter, we hope to discuss data suggesting that metals may be important also to the structure of certain single-chain proteins and to the general phenomenon of stabilization of protein structure. 

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... 

The above principle is amply illustrated by the small molecule systems discussed in Section III. The roles proposed for zinc ion in the three enzyme systems discussed in Section IV also adhere to this principle. The accumulated experimental evidence makes it highly probable that zinc ion has a Lewis acid catalytic function both in the horse liver alcohol dehydrogenase-catalyzed reduction of aldehydes, and in the carboxypeptidase A-catalyzed hydrolysis of peptides. In contrast, the accumulated experimental evidence supports a role for zinc ion involving the enhancement of water nucleophilicity via inner sphere coordination in the carbonic anhydrase-catalyzed hydration of CO 2. The substrates for... 

Mitochondrial malate dehydrogenase (MDH) from several species has been shown to exist in several enzymically active forms which also appear to be conformational isoenzymes (Kitto et al, 1966, 1970). Kitto et al (1970) showed, in contrast to Epstein and Schechter (1968), that these MDH s were interconvertible in vitro, had the same amino acid compositions and molecular weights, but differed, once reversibly denatured, considerably in their heat stability. Similar interconversion of isoenzymes has also been observed with purified preparations of horse liver alcohol dehydrogenase (Lutstorf and von Wartburg, 1969). The question arises if such conformers are of any physiological or functional significance. It is possible that beeause of their differences in surface charge, the various conformative isoenzymes are differently bound within a cell. 

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])...

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