Horse liver alcohol

Eklund, H., et al. Three-dimensional structure of horse liver alcohol dehydrogenase at 2.4 A resolution. 

ADH Horse liver alcohol dehydrogenase, an enzyme dimer of identical 374 amino acid polypeptide chains. The amino acid composition of ADH is reasonably representative of die norm for water-solnble proteins. 

Figure 8.27 Reduction of aldehyde in SCCO2 by an isolated enzyme, horse liver alcohol dehydrogenase (HLADH) [20c] (a) Reaction scheme (b) fluorinated coenzyme soluble in CO2 and (c) effect of coenzyme on the reaction.

Figure 17.8 Catal3ftic zinc center of horse liver alcohol dehydrogenase revealed from an X-ray crystallographic structure (PDB file 20HX) [Al-Karadaghi et al., 1994]. The bound NADH cofactor, a molecule of the inhibitor dimethylsulfoxide (DMSO), and the amino acid residues that coordinate the Zn are shown as sticks shaded according to the elements, and the Zn center is shown as a gray sphere, while the protein is shown in thin gray lines.

Diol bonded silica Glucosamine, bovine serum albumin, immunoglobulin, acetylcholine esterase, horse liver alcohol dehydrogenase [136]... 

Fig. 7. Horse-liver alcohol dehydrogenase (HLADH) catalyzed alcohol oxidation at a graphite felt anode modified by poly(acrylic acid) (PAA) under coimmobilization of ferrocene derivatives (Fc), diaphorase (Dp), and HLADH [39]...

We then coupled the regeneration system 1 to the horse liver alcohol dehydrogenase (HLADH) catalyzed oxidation of cyclohexanol to cyclohexanone as a model system (Fig. 9). 

Recently, we adopted the same system for the reduction of 4-phenyl-2-butanone to (S)-4-phenyl-2-butanol using the NADH-dependent horse liver alcohol dehydrogenase (HLADH) and S-ADH from Rhodococcus sp [68] with high enantioselectivity (Fig. 17) [69]. As mediator, we applied the low-molecular... 

Phosphorescence and ODMR are additional spectroscopies that can be used to investigate intramolecular interactions that affect tyrosine residues in proteins and polypeptides/215,216) An example is tyrosine and tyrosinate in horse liver alcohol dehydrogenase.(202) The same approach has been used to study the role of tyrosine in the mechanism of action of carboxypeptidase B.(21/,218) jn botli these proteins, as in other proteins which contain both... 

In the case of horse liver alcohol dehydrogenase, a homodimeric enzyme, Subramanian et al.(202) used the relative phosphorescence of tyrosine and tryptophan to examine the effects of various ternary complexes known to selectively quench the fluorescence of the tryptophans of each subunit. One proposed quenching mechanism is the formation of a ground-state tyrosinate in a ternary complex at neutral pH.(201) This tyrosinate, by being a resonance... 

For horse liver alcohol dehydrogenase, denaturation by guanidine hydrochloride resulted in a decrease in phosphorescence lifetime parallel with loss of activity.(79) With urea as a denaturant, the decrease in phosphorescence lifetime appeared cooperative, and it is suggested that the denaturant loosened intramolecular interactions (such as hydrogen bonds), resulting in greater fluidity of the tryptophan environment.(80)... 

J. A. B. Ross, C. J. Schmidt, and L. Brand, Time-resolved fluorescence of the two tryptophans in horse liver alcohol dehydrogenase, Biochemistry 20, 4369-4377 (1981). 

Yeast alcohol dehydrogenase (YADH), catalysis of reduction by NADH of acetone formate dehydrogenase (FDH), oxidation by NAD of formate horse-liver alcohol dehydrogenase (HLAD), catalysis of reduction by NADH of cyclohexanone With label in NADH, the secondary KIE is 1.38 for reduction of acetone (YADH) with label in NAD, the secondary KIE is 1.22 for oxidation of formate (FDH) with label in NADH, the secondary KIE is 1.50 for reduction of cyclohexanone (HLAD). The exalted secondary isotope effects were suggested to originate in reaction-coordinate motion of the secondary center. 

In the following year, Cleland and his coworkers reported further and more emphatic examples of the phenomenon of exaltation of the a-secondary isotope effects in enzymic hydride-transfer reactions. The cases shown in Table 1 for their studies of yeast alcohol dehydrogenase and horse-liver alcohol dehydrogenase would have been expected on traditional grounds to show kinetic isotope effects between 1.00 and 1.13 but in fact values of 1.38 and 1.50 were found. Even more impressively, the oxidation of formate by NAD was expected to exhibit an isotope effect between 1.00 and 1/1.13 = 0.89 - an inverse isotope effect because NAD" was being converted to NADH. The observed value was 1.22, normal rather than inverse. Again the model of coupled motion, with a citation to Kurz and Frieden, was invoked to interpret the findings. 

Horse liver alcohol dehydrogenase and the F93W mutant, hydride transfer from henzyl alcohol to NAD in MeOH/water. 

Horse liver alcohol dehydrogenase, F93W mutant with 1224 also mutated to G,A,V,L. hydride transfer from benzyl alcohol to NAD Heterotetrameric sarcosine oxidase of Arthrobacter sp. 1-IN, proton transfer from adduct of FAD with sarcosine-(CH3) and sarcosine-(CD3)... 

Two studies in Table 2 (entries 8 and 9) proceed from previous reports question of tunnehng in the action of horse-liver alcohol dehydrogenase. 

Fig. 6 Illustration from Chin and Klinman. Increased catalytic activity of horse-liver alcohol dehydrogenase in the oxidation of benzyl alcohol to benzaldehyde by NAD, measured by cat/ M (ordinate), correlates with the Swain-Schaad exponent for the -secondary isotope effect (abscissa), for which values above about four are indicators of tunneling. This is a direct test of the hypothesis that tunneling in the action of this enzyme contributes to catalysis. As the rate increases by over two orders of magnitude and then levels off, the anomalous Swain-Schaad exponents also increase and then level off. Reproduced from Ref. 28 with the permission of the American Chemical Society.

Chin, J.K. and Klinman, J.P. (2000). Probes of a role for remote binding interactions on hydrogen tunneling in the horse liver alcohol dehydrogenase reaction. Biochemistry 39, 1278-1284... 

However, a pure enzyme, like horse liver alcohol dehydrogenase (HLADH), shows not only high stereoselectivity but regioselectivity as well, affording, for example, 89% yield of monoalcohol 5 from dione 4 with ee higher than 99% [14]. 

The latter were transformed into (+)-64, (-)-65 and (+)-66, respectively, in good yields. Lactone (+)-66 was also prepared in 37 % yield by stereospecific Horse Liver Alcohol Dehydrogenase (HLADH) catalyzed oxidation of the meso-diol 67. Lactones (-)-64 and (+)-64 are potentially interesting starting materials for the... 

Figure 9. Preparation of L- and D-lactaldehyde and L- and D-2-hydroxybutyraldehyde. HLADH, horse liver alcohol dehydrogenase (NADH was regenerated by glucose/glucose dehydrogenase).

All the enzymes used in the work described above are quite stable at room temperature and can be used in a free form. They can also be used in an immobilized form to improve the stability and to facilitate the recovery. Many immobilization techniques are available today (25). The recent procedure developed by Whitesides et al using water-insoluble, cross-linked poly(aerylamide-acryloxysuccinimide) appears to be very useful and applicable to many enzymes (37). We have found that the non-crosslinked polymer can be used directly for immobilization in the absence of the diamine cross-linking reagent. Reaction of an enzyme with the reactive polymer produces an immobilized enzyme which is soluble in aqueous solutions but insoluble in organic solvents. Many enzymes have been immobilized by this way and the stability of each enzyme is enhanced by a factor of greater than 100. Horse liver alcohol dehydrogenase and FDP aldolase, for example, have been successfully immobilized and showed a marked increase in stability. 

The enzyme chemistry of cyclopropylmethanols has been studied both as inhibitors and mechanistic probes [4, 47]. Thus, a series of alkylcyclopropyl-methanol derivatives have been proved as being inhibitors of horse liver alcohol dehydrogenase. There are two sites in the cyclopropylmethanol inhibitors able of reacting with nucleophiles ... 

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