Clinical Box 31 Alcohol Sensitivity in East Asian Populations

The majority of beverage-derived ethanol is metabolized by two enzyme systems in the liver: alcohol dehydrogenase and aldehyde dehydrogenase.


Fthanol l~ Acetaldehyde I Acetate

Alcohol Dehydrogenase Aldehyde Dehydrogenase

Both enzymes utilize a coenzyme, NAD+, as the electron acceptor in order to catalyze the oxidation of their substrates. Humans possess multiple forms of both enzymes; these are termed isoenzymes. Isoenzymes are related gene products that catalyze similar reactions, but do so with differing affinities for substrates and/or with differing rates of catalysis. A common occurrence among individuals with East Asian ancestry (China, Japan, North and South Korea) is intolerance toward the consumption of alcoholic beverages. This ethanol intolerance is characterized by a rapid "flushing reaction" in which vasodilation of the blood vessels in the face gives rise to a reddened appearance within a few minutes of ethanol consumption. In addition to this external symptom, susceptible individuals also experience nausea, dizziness (syncope), and rapid heart rates (tachycardia) following the consumption of alcoholic beverages (Hurley, Edenberg and Li, 2002).

The primary basis for these reactions is the presence of a different form of aldehyde dehydrogenase, termed a polymorphic variant, in this ethnic population. The polymorphic variant of this enzyme is the result of a single nucleotide exchange in the gene that gives rise to a single amino acid change in the enzyme structure. This amino acid substitution decreases the ability of aldehyde dehydrogenase to use its coenzyme NAD+ by 200-fold. We measure this change by reporting the concentration of NAD+ that gives half-maximal activity—the KM value—which, in this case, requires that we use 200 times the concentration we would use for the enzyme without this mutation.

At the same time, this mutation also decreases the rate at which the enzyme can catalyze its reaction by tenfold. As a consequence, this less active form of the enzyme has less than 10% of the activity of the more active form under the conditions that exist in the cell. The human liver cell maintains a relatively constant concentration of NAD near 0.5 X 10 3 M. However, due to this amino acid change, the less active form of aldehyde dehydrogenase would require more than 100 times this concentration to reach maximal activity, which would still be tenfold lower than the more active form.

The basis for the physiological reaction to ethanol consumption is that the intracellular levels of acetaldehyde begin to rise as a consequence of the poor catalytic properties of this enzyme and spill out into the serum, where the acetaldehyde causes vasodilation, dizziness, and nausea. Ultimately, another enzyme takes over the metabolism of the acetaldehyde produced by alcohol dehydrogenase. However, the levels of acetaldehyde must accumulate before this enzyme can effectively utilize acetaldehyde as a substrate (it has a high KM for acetaldehyde), and this accumulation of acetaldehyde is what leads to the aversive reaction to beverage ethanol consumption.

Ping-pong sequential Bi Bi reaction mechanism. In a ping-pong sequential mechanism, the substrate A is chemically converted to P, and the enzyme, E, is modified in some manner by this reaction to become F. Then substrate B binds to this modified form of the

enzyme, regenerates the original form of the enzyme, E, and is converted to the product Q in this process. Classically, the enzymes that catalyze the transfer of amino groups from one amino acid to a different alpha-ketoacid (termed transaminases) obey ping-pong mechanisms. A transaminase utilizes the coenzyme pyridoxal phosphate to pick up the amine group from one amino acid, for instance aspartate, to generate the alpha-ketoacid oxaloacetate. Coenzymes are small molecules that are essential for the normal activity of the enzyme. During the transamination process, the coenzyme pyridoxal phosphate is chemically modified to pyridoxamine phosphate, which remains tightly bound to the enzyme active site. In the next step, a second alpha-ketoacid, for instance alpha-ketoglutarate, binds to this modified form of the coenzyme and picks up the amino group from pyridoxamine phosphate to regenerate the pyridoxal phosphate in the active site and produce the second product, glutamate.

Random Bi Bi reaction mechanism.


In a random mechanism, the enzyme still goes through a "ternary complex" as observed in the ordered-sequential situation, but the substrates can bind in either order and the products can dissociate in either order. In this case, there is no structural or chemical pressure to select for preferential binding and release of the ligands to the enzyme surface as occurs in the ordered-sequential and ping-pong sequential mechanisms.

Kinetic expressions for Bi-reactant systems Ordered Bi Bi mechanism:

For this enzymatic mechanism, the complete velocity expression, in the absence of P and Q (initial rate, steady-state assumptions), is as follows:

These kinetic constants are defined in Eqs. (3.22) to (3.24) in terms of their individual rate constants; kp and k_p are the forward and reverse "product forming" rate constants.

KiA = 1 (true dissociation constant for substate A) (3.22) ki

As you can see, the complexity of the kinetic expression increases tremendously as does the interpretation of the physical meaning of the individual kinetic constants. However, the practical descriptions for KM, kcat, and kcat/KM outlined in Sec. 3.1.2 still apply here and serve as a basis for understanding what these more complex Michaelis constants actually describe in these enzymatic systems. However, due to their increased complexity, the kinetic experiment that properly determines the kinetic constants of multisubstrate enzymes becomes two-dimensional. This means that the concentration of both substrates must be varied simultaneously. Practically, this is carried out by varying the concentration of one substrate at various constant concentrations of the second. The kinetic plots are thus a series of saturation curves, in which the apparent saturation for one substrate is plotted at various concentrations of the second substrate (Fig. 3.5). Enzymatic mechanisms that proceed through a ternary complex show a set of intersecting lines when plotted using the method of Lineweaver and Burk. This is because the apparent saturation level of one substrate influences the apparent saturation level of the other substrate and vice versa.

Ping-pong sequential mechanism:

The complete velocity expression for the formation of P and Q from A and B for this enzymatic mechanism is as follows; for simplicity we have omitted the rate constants from these relationships.

Figure 3.5 Ordered Bi Bi Lineweaver-Burk plot. The double reciprocal (Lineweaver-Burk) plot shows the results of a covary kinetic experiment for a ordered Bi Bi kinetic system. In this plot, each line represents a single fixed concentration of the second substrate, B. The reciprocal of the velocities obtained at this concentration of B and at varying concentrations of A are plotted against the reciprocal of the concentration of A that gave rise to the observed velocity.

Figure 3.5 Ordered Bi Bi Lineweaver-Burk plot. The double reciprocal (Lineweaver-Burk) plot shows the results of a covary kinetic experiment for a ordered Bi Bi kinetic system. In this plot, each line represents a single fixed concentration of the second substrate, B. The reciprocal of the velocities obtained at this concentration of B and at varying concentrations of A are plotted against the reciprocal of the concentration of A that gave rise to the observed velocity.

Figure 3.6 Ping pong Lineweaver-Burk plot. The double reciprocal (Lineweaver-Burk) plot for a ping-pong reaction mechanism results from a series of experiments with fixed concentrations for substrate B at varying concentrations of A.

As in the ordered mechanism, the proper way to evaluate the kinetic constants for enzymes that obey this mechanism is to vary both substrates simultaneously and to thereby obtain a set of activity versus substrate concentration curves. However, unlike enzyme mechanisms that proceed through a ternary complex, ping-pong mechanisms do not show interdependence of substrate binding. Consequently, plotting the data using the Lineweaver-Burk equation yields a set of parallel lines with constant slope values and differing x- and y-intercepts (Fig. 3.6). This is because substrate binding to the different enzyme forms (E or F) influences only the maximal velocity that can be obtained, but does not affect the apparent affinity of the different enzyme forms for the second substrate.

The reader is referred to other textbooks and references for information on how to obtain the relevant kinetic constants from these covariation experiments.

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