Enzyme horse liver

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.

JB Jones. An illustrative example of a synthetically useful enzyme horse liver alcohol dehydrogenase. In R Porter, S Clark eds. Enzymes in Organic Synthesis. London Pitman Publishing, 1985, pp 3-21. 

Previously, Beckman and co-workers had prepared nicotinamide adenine dinucleotide (NAD) with a fluorophilic ponytail (FNAD). This molecule was able to act as an affinity surfactant and extract the enzyme horse liver alcohol dehydrogenase (HLADH) from an aqueous medium into methoxynona-fluorobutane (HFE) (Figure 7.24). Interestingly, the addition of potential... 

Panza et al. synthesized a C02-philic amphiphile from the coenzyme nicatinamide adenine dinucleotide (MW 664) and a covalently attached perfluoropolyether (MW 2500) (Figure 7B) (73). The fluorofunctional coenzyme (FNAD) was soluble up to 5 mM in CO2 at room temperature and 1400 psi. The C02-soluble FNAD was able to participate in a cyclic oxidation/reduction reaction catalyzed by the enzyme horse liver alcohol dehydrogenase (HLADH) in CO2 at room temperature and 2600 psi. 

In a second synthesis of labeled phenylalanine Battersby et al. (305, 306) used the pro-R specific enzyme horse liver alcohol dehydrogenase to reduce [ HJbenzaldehyde 306, Ha = H. The (lS)-[l- H,]benzyl alcohol 307 obtained was converted via tosylation and reaction with malonate anion to the acid 308 (Scheme 81), which, on bromination and ammonolysis, gave (2RS, 3R)-[3- Hi]phenylalanine 297, He = H. Ife and Haslam (309) used the more direct replacement of the tosylates of the alcohol 307 with acetamidomalonic ester in a similar synthesis of (3R)- and (3S)-[3- Hi]pheny-lalanines. Fermenting yeast replaced liver alcohol dehydrogenase in a further synthesis (310). 

Fig. 13. Phosphorescence emission of the enzyme horse liver alcohol dehydrogenase at 1.3 K in 50 % ethylene glycol-water showing the optically-resolved origins of the two tryptophan sites of the enzyme. Excitation is at 295 nm. (From Zuclich et al. 1 )...

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. 

Laufberger had tried to obtain the protein from horse liver, but it did not crystallize, and as he described to me when I met him in Prague some years ago, in those days everyone wanted to have protein crystals as a criteria of purity. Although James Sumner had crystallized jack bean urease in 1926, his preparations were somewhat impure, and it was only in the mid-1930s, when John Northrop and Moses Kubnitz showed that there is a direct correlation between the enzymatic activities of crystalline pepsin, trypsin and chymotrypsin that the protein nature of enzymes was generally accepted. 

Using two types of specially synthesized rhodium-complexes (12a/12b), pyruvate is chemically hydrogenated to produce racemic lactate. Within the mixture, both a d- and L-specific lactate dehydrogenase (d-/l-LDH) are co-immobilized, which oxidize the lactate back to pyruvate while reducing NAD+ to NADH (Scheme 43.4). The reduced cofactor is then used by the producing enzyme (ADH from horse liver, HL-ADH), to reduce a ketone to an alcohol. Two examples have been examined. The first example is the reduction of cyclohexanone to cyclohexanol, which proceeded to 100% conversion after 8 days, resulting in total TONs (TTNs) of 1500 for the Rh-complexes 12 and 50 for NAD. The second example concerns the reduction of ( )-2-norbornanone to 72% endo-norbor-nanol (38% ee) and 28% exo-norbornanol (>99% ee), which was also completed in 8 days, and resulted in the same TTNs as for the first case. 

The enzymes used by these workers were cholinesterase, prepared from horse serum, and horse-liver esterase. Parallel experiments were carried out with twice crystallized ovalbumin, and with an aged, dialysed specimen of horse serum with negligible esterase activity. 

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... 

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. 

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.

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]. 

It is noteworthy, here, that one conunercial source of purified alcohol dehydrogenase is horse liver. There is a considerable quantity of this enzyme in horse liver. It is likely that alcohol arises from fermentation in the caecum of the high content of fibre present in the food of the horse. Hence its liver will be exposed chronically to high concentrations of alcohol, and the liver adapts by synthesising large quantities of the enzyme. 

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 ... 

This zinc metalloenzyme [EC 1.1.1.1 and EC 1.1.1.2] catalyzes the reversible oxidation of a broad spectrum of alcohol substrates and reduction of aldehyde substrates, usually with NAD+ as a coenzyme. The yeast and horse liver enzymes are probably the most extensively characterized oxidoreductases with respect to the reaction mechanism. Only one of two zinc ions is catalytically important, and the general mechanistic properties of the yeast and liver enzymes are similar, but not identical. Alcohol dehydrogenase can be regarded as a model enzyme system for the exploration of hydrogen kinetic isotope effects. 

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... 

AOT/isooctane/ buffer Horse liver alcohol dehydrogenase Microemulsion system that is temperature sensitive to phase separate was used for recovery of proteins and enzymes [283]... 

Horse liver alcohol dehydrogenase (HLADH (E.C. 1.1.1.1), commercially available) is a well-documented enzyme capable of catalyzing the enantioselective oxidation of acyclic and cyclic meso-configurated dimethanol derivatives to chiral lactols and further to the corresponding chiral lactones with high enantioselectivity and in high yield (Table 11) 162 ,69. Incases where the two enantiomeric lactols are formed, a kinetic enantiomer separation can occur in the second oxidation step166. 

Horse liver alcohol dehydrogenase is able to oxidise primary alcohols—except methanol—and to reduce a large number of aldehydes. Aqueous solution or organic solvents can be used [62]. As there are no new developments concerning this enzyme, the reader is referred to the review of Schreier [1]. 

The reduction of 3- and 4-thiepanones (41 and 42) was reported using either hydride (LAH) (67AG(E)872, 70JOC584) or horse liver alcohol dehydrogenase enzymes which gave the 3-hydroxy- (136) and 4-hydroxy- (43) thiepane in optically active form (81CJC1574. ... 

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). 

Different purified or partially purified enzymes were tested successfully, such as horse liver dehydrogenase [3], Sulfolobus solfataricus dehydrogenase [4], Pischia pastoris alcohol oxidase [5, 6], the baker s yeast alcohol dehydrogenase [7], and finally lipolytic enzymes, which probably constitute the major part of the work devoted to the use of enzymes working at the solid/gas interface, as summarized in a recent publication [8]. 

Eklund et al. suggested that the side chains of Ser 48 and His 51 act as a proton relay system to remove the proton from the alcohol, in step b of Eq. 15-7, leaving the transient zinc-bound alcoholate ion, which can then transfer a hydride ion to NAD+, in step c.52 The shaded hydrogen atom leaves as H+. The role of His 51 as a base is supported by studies of the inactivation of the horse liver enzyme by diethyl pyrocarbonate57 and by directed mutation of yeast and liver enzymes. When His 51 was substituted by Gin the pKa of 7 was abolished and the activity was decreased ten-fold.58... 

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