Alcohol dehydrogenase coenzyme specificity

The dehydrogenases discussed in this section catalyze the oxidation of alcohols to carbonyl compounds. They utilize either NAD+ or NADP+ as coenzymes. The complex of the enzyme and coenzyme is termed the holoenzyme the free enzyme is called the apotnzyme. Some dehydrogenases are specific for just one of the coenzymes a few use both. The reactions are readily reversible, so that carbonyl compounds may be reduced by NADH or NADPH. The rates of reaction in either direction are conveniently measured by the appearance or disappearance of the reduced coenzyme, since it has a characteristic ultraviolet absorbance at 340 nm. The reduced coenzymes also fluoresce when they are excited at 340 nm, which provides an even more sensitive means of assay. 

The alcohol dehydrogenases are zinc metalloenzymes of broad specificity. They oxidize a wide range of aliphatic and aromatic alcohols to their corresponding aldehydes and ketones, using NAD+ as a coenzyme (see equation 16.1). The two most studied enzymes are those from yeast and horse liver. The crystal structures... 

The biochemical characterization of several alcohol dehydrogenases and their exploitation potential demonstrate that these enzymes are most important tools for biochemists. Amino acid sequences of several ADFls are available so far, and alignment studies allow to establish ADH families and to consider their probable evolutionary relationships. For preparative applications, however, particular properties of an enzyme are essential prerequisites, such as enzyme stability and availability, its substrate specificity, or reaction selectivity. Enzymes with NAD as coenzyme are clearly preferred to NADP-dependent ones in practice, because NAD has a significantly higher stability [186-188], a lower price and, is in general, easier to regenerate. 

Enzyme Cofactors- In many enzymatic reactions, and in particular biological reactions, a second substrate (i.e., species) must be introduced to activate the enzyme. This substrate, which is referred to as a cofactor or coenzyme even though it is not an enzyme as such, attaches to the enzyme and is most often either reduced or oxidized during the course of die reaction. The enzyme-cofactor complex is referred to as a holoenzyme. The inactive form of the enzyme-cofactor complex for a specific reaction and reaction direction is called an apoenzyme. An example of the type of system in which a cofactor is used is the formation of ethanol from acetaldehyde in the presence of the enzyme alcohol dehydrogenase (ADH) and the cofactor nicotinamide adenine dinuoleotide (NAD) ... 

The substrate specificities of the alcohol dehydrogenases from Pseudomonas sp. strain PED and Lactobacillus kefir have been investigated. It was reported that they reduce wide varieties of ketonesI6- 7l Both reactions use 2-propanol for the regeneration of coenzyme and produce (ft)-alcohols as depicted in Table 15-12. However, they require different coenzymes. The alcohol dehydrogenase from the Pseudomonas sp. uses NADH and transfers to pro-ft hydride of NADH to the si-face of carbonyl compounds as shown in Sect. 15.1.1.1. The mechanism is ordered bi-bi with the coenzyme binding first and released last. On the other hand, the enzyme from... 

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.

It would be well to point out a few examples which illustrate the overlap of asymmetric reduction studies and molecular biochemistry. Diphosphopyridine nucleotide (DPN) and triphospho-pyridine nucleotide (TPN) are important coenzymes in biochemical oxidation reduction reactions. Certain enzymes function as catalysts for the reversible transfer of hydrogen between these nucleotides and a substrate for which the enzyme is specific. For example, DPN and the enzyme, alcohol dehydrogenase (ADH), form a redox system with ethanol. Using deuterium labeled reducing agent and substrate, Westheimer, Vennesland,... 

Coenzyme Specificity of Dehydrogenases. A number of dehydrogenases have been reported to require DPN, others are specific for TPN, and several react with both, although not necessarily at the same rate. Examples have already been cited of enzymes that catalyze identical reactions but possess different coenzyme requirements. An additional means for studying pyridine nucleotide reactions became available with desamino DPN. Certain dehydrogenases (e.g., liver alcohol dehydrogenase) react at equal rates with DPN and desamino DPN. Others (as... 

We have emphasized the specific character of macromolecules. Even in the arsenal of enzymes common to all cells we find indications of this specificity. The glucose dehydrogenase of vertebrate liver for example, is not inhibited by toluene, whilst that of E. colt is inhibited. Yeast alcohol dehydrogenase is completely inhibited by O-OOIM iodoacetate, whilst even at a concentration of O-OIM, animal alcohol dehydrogenase remains imaffected. Glutamic add dehydrogenase of yeast requires TPN as a coenzyme whilst the same enzyme from plants needs DPN. 

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