Cooperative Behavior in Enzymes

Many enzymes are classified as cooperative enzymes. A common feature of all cooperative enzymes is that they contain multiple copies of the same polypeptide chain to form the functional enzyme, or a set of different polypeptide chains combine to form the functional enzyme. A consequence of these multiple polypeptide chains is that there are multiple active sites that all catalyze the same reaction. Not all enzymes with multiple active sites show cooperativity, but all cooperative enzymes have multiple active sites. In contrast to the enzymes in the preceding section, cooperative enzymes communicate information between their active sites. This cooperativity could result in either the activation of the other active site(s) (positive cooperativity) or the inhibition of the other active site(s) (negative cooperativity). Many cooperative enzymes also contain binding sites for other ligands (regulatory molecules) that influence the nature or degree of cooperativity between the active sites in the enzyme complex.

The enzyme aspartate transcarbamoylase is a cooperative enzyme that shows allosteric regulation by components of the metabolic pathway in which it participates. This enzyme catalyzes the first step in the biosynthetic

Figure 3.12 Feedback inhibition. The simplified diagram of the synthetic pathway for CTP shows the feedback inhibition of CTP on the first step in the pathway which is catalyzed by aspartate transcarb-amoylase.

Figure 3.12 Feedback inhibition. The simplified diagram of the synthetic pathway for CTP shows the feedback inhibition of CTP on the first step in the pathway which is catalyzed by aspartate transcarb-amoylase.

pathway for production of cytidine triphosphate (CTP) (Fig. 3.12). CTP is an essential nucleotide triphosphate for a number of metabolic reactions.

One of the more important uses of CTP is during the duplication of the cell's genetic material prior to mitosis. Consequently, prior to cell division there is a large requirement for CTP for DNA synthesis, but that requirement diminishes when DNA synthesis is not proceeding. To address this variable metabolic demand, the cell needs to sense when sufficient resources are available for CTP synthesis and when enough CTP has been synthesized for its current metabolic needs. In this regard, the enzyme is allosterically activated by adenosine triphosphate (ATP). Since ATP is required for the reaction catalyzed by this enzyme, a threshold level of ATP must be present in order to activate the enzyme. In contrast, the eventual product of the metabolic pathway, CTP, is an allosteric inhibitor of aspartate transcarbamoylase such that when sufficient levels of CTP are available the reaction is inhibited (Fig. 3.13).

These allosteric regulatory molecules bind to sites on the enzyme surface that are distinct from the active site (there is a separate ATP binding site in the active site for catalyzing the actual reaction). When molecules bind to these sites, conformational changes occur that influence the ability of the active sites to catalyze the reaction. The regulation of enzyme activity within a metabolic pathway by the pathway's ultimate product is termed feedback inhibition. The end product of the pathway controls the flux through the pathway, preventing too much product synthesis, which would be energetically wasteful.

Notice that the substrate saturation profiles in Fig. 3.13 are not hyperbolic. Cooperative enzymes show sigmoidal (S-shaped) saturation curves that reflect the fact that initial binding of a substrate to one active site is difficult, but its binding activates the other active sites for binding the substrates. The saturation kinetics for cooperative enzymes is governed by the following equation.

[Aspartate]

Figure 3.13 Sigmoidal substrate binding curves. The center curve is the substrate saturation curve for the enzyme aspartate transcarbamoylase. This saturation curve is shifted to the left (making the enzyme more active at any given concentration of aspartate) in the presence of the allosteric activator ATP. It is shifted to the right (making the enzyme less active at any given concentration of aspartate) in the presence of the allosteric inhibitor CTP.

[Aspartate]

Figure 3.13 Sigmoidal substrate binding curves. The center curve is the substrate saturation curve for the enzyme aspartate transcarbamoylase. This saturation curve is shifted to the left (making the enzyme more active at any given concentration of aspartate) in the presence of the allosteric activator ATP. It is shifted to the right (making the enzyme less active at any given concentration of aspartate) in the presence of the allosteric inhibitor CTP.

The exponent, or Hill coefficient, n, is indicative of the type and degree of cooperativity. If n = 1, then standard Michaelis-Menton kinetics are observed; if n >1, then the enzyme exhibits positive cooperativity; if n <1, then the enzyme exhibits negative cooperativity. In order to determine the value of n in this equation, we generally perform a separate analysis of the data and fit the data obtained to the Hill equation [Eq. (3.35)]. A Hill plot is generated by plotting the logarithm of the fraction of enzyme with bound substrate divided by the fraction of enzyme free in solution versus the logarithm of the substrate concentration. This relationship produces a linear relationship in which the slope of the line in the middle of the plot is equal to n. In this equation, the quantity y is the fraction of enzyme to which substrate is bound.

In the case of positive cooperativity, the maximal value for n is equal to the number of substrate binding sites in the enzyme complex, though this value does not always reach this limit. For instance, hemoglobin binds oxygen molecules cooperatively to the four binding sites present in this tetrameric protein molecule (tetramer = four copies), but its Hill coefficient is only 2.9.

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