Irreversible Inhibition

Heme

Binds ions, O2, and electrons; contains iron cofactor

Flavin

Binds electrons

Retinal

Converts light energy

► Prosthetic groups. These distinctive molecular groupings are permanently bound to their enzymes. They include the heme groups that are attached to the oxygen-carrying protein hemoglobin (shown in Figure 3.8).

Coenzymes are like substrates in that they are not permanently bound to the enzyme, and must collide with the enzyme and bind to its active site. A coenzyme can be consid-

Enzyme Confactor Illustration
6.15 An Enzyme with a Coenzyme Some enzymes require coenzymes in order to function. This illustration shows the relative sizes of the four subunits (red, orange, green, and purple) of the enzyme glyceraldehyde 3-phosphate dehydrogenase and its coenzyme, NAD (white).

ered a substrate because it changes chemically during the reaction and then separates from the enzyme to participate in other reactions. Coenzymes move from enzyme molecule to enzyme molecule, adding or removing chemical groups from the substrate.

ATP and ADP can be considered coenzymes because they are necessary for some reactions, are changed by reactions, and bind to and detach from the enzyme. In the next chapter, we will encounter coenzymes that function in energy processing by accepting or donating electrons or hydrogen atoms. In animals, some coenzymes are produced from vitamins that must be obtained from food—they cannot be synthesized by the body. For example, the B vitamin niacin is used to make the coenzyme NAD.

Substrate concentration affects reaction rate

For a reaction of the type A ^ B, the rate of the uncatalyzed reaction is directly proportional to the concentration of A (Figure 6.16). The higher the concentration of substrate, the more reactions per unit of time. Addition of the appropriate enzyme speeds up the reaction, of course, but it also changes the shape of the plot of rate versus substrate concentration. At first, the rate of the enzyme-catalyzed reaction increases as the substrate concentration increases, but then it levels off. When further increases in the substrate concentration do not significantly increase the reaction rate, the maximum rate is attained.

Since the concentration of an enzyme is usually much lower than that of its substrate, what we are seeing is a saturation phenomenon like the one that occurs in facilitated diffusion (see Chapter 5). When all the enzyme molecules are bound to substrate molecules, the enzyme is working as fast as it can—at its maximum rate. Nothing is gained by adding more substrate, because no free enzyme molecules are left to act as catalysts.

Concentration of substrate

6.16 Catalyzed Reactions Reach a Maximum Rate Because there is usually less enzyme than substrate present, the reaction rate levels off when the enzyme becomes saturated.

Concentration of substrate

6.16 Catalyzed Reactions Reach a Maximum Rate Because there is usually less enzyme than substrate present, the reaction rate levels off when the enzyme becomes saturated.

The maximum rate of an enzyme reaction can be used to measure how efficient the enzyme can be—that is, how many molecules of substrate are converted to product per unit of time when there is an excess of substrate present. This turnover number ranges from 1 molecule every 2 seconds for lysozyme (see Figure 6.13) to an amazing 40 million molecules per second for the liver enzyme catalase.

Metabolism and the Regulation ofEnzymes

A major characteristic of life is homeostasis, the maintenance of stable internal conditions. Regulation of the rates at which our thousands of different enzymes operate contributes to metabolic homeostasis. In the remainder of this chapter, we will investigate the role of enzymes in organizing and regulating metabolism. In living cells, the activity of enzymes can be activated or inhibited in various ways, so the presence of an enzyme does not necessarily ensure that it is functioning. There are mechanisms that alter the rate at which some enzymes catalyze reactions, making enzymes the target points at which entire sequences of chemical reactions can be regulated. Finally, we examine how the environment—namely, temperature and pH—affects enzyme activity.

Metabolism is organized into pathways

An organism's metabolism is the totality of the biochemical reactions that take place within it. Metabolism transforms raw materials and stored potential energy into forms that can be used by living cells. Metabolism consists of sequences of enzyme-catalyzed chemical reactions called pathways. In these sequences, the product of one reaction is the substrate for the next:

Some metabolic pathways are anabolic, synthesizing the important chemical building blocks from which macro-molecules are built. Others are catabolic, breaking down molecules for usable free energy, recycling monomers, or inactivating toxic substances. The balance among these anabolic and catabolic pathways may change depending on the cell's (and the organism's) needs. So a cell must regulate all its metabolic pathways constantly.

Enzyme activity is subject to regulation by inhibitors

Various inhibitors can bind to enzymes, slowing down the rates of enzyme-catalyzed reactions. Some inhibitors occur naturally in cells; others are artificial. Naturally occurring inhibitors regulate metabolism; artificial ones can be used to treat disease, to kill pests, or in the laboratory to study how enzymes work. Some inhibitors irreversibly inhibit the enzyme by permanently binding to it. Others have reversible effects; that is, they can become unbound from the enzyme. The removal of a natural reversible inhibitor increases an enzyme's rate of catalysis.

irreversible inhibition. Some inhibitors covalently bond to certain side chains at the active sites of an enzyme, thereby permanently inactivating the enzyme by destroying its capacity to interact with its normal substrate. At the beginning of this chapter we described aspirin, which adds an acetyl group to a serine residue at the active site of cyclo-oxygenase, preventing this serine from taking part in chemical catalysis.

Another example of an irreversible inhibitor is DIPF (di-isopropylphosphorofluoridate), which also reacts with serine (Figure 6.17). DIPF is an irreversible inhibitor of acetyl-cholinesterase, an enzyme that is essential for the orderly propagation of impulses from one nerve cell to another. Because of their effect on acetylcholinesterase, DIPF and other similar compounds are classified as nerve gases. One of them, Sarin, was used in an attack on the Tokyo subway in 1995, resulting in a dozen deaths and hundreds hospitalized. The widely used insecticide malathion is a derivative of DIPF that inhibits only insect acetylcholinesterase, not the mammalian enzyme.

reversible inhibition. Not all inhibition is irreversible. Some inhibitors are similar enough to a particular enzyme's natural substrate to bind noncovalently to its active site, yet different enough that the enzyme catalyzes no chemical reaction. While such a molecule is bound to the enzyme,

Active site

Irreversible Inhibition

DIPF, an irreversible inhibitor, reacts with the hydroxyl group of serine.

Active site

Irreversible Inhibition

Covalent attachment of DIPF to the active site prevents substrate from entering, thus disabling the enzyme.

DIPF, an irreversible inhibitor, reacts with the hydroxyl group of serine.

Covalent attachment of DIPF to the active site prevents substrate from entering, thus disabling the enzyme.

6.17 Irreversible Inhibition DIPF forms a stable covalent bond with the side chain of the amino acid serine at the active site of the enzyme trypsin.

the natural substrate cannot enter the active site; thus, the inhibitor effectively wastes the enzyme's time, preventing its catalytic action. Such molecules are called competitive inhibitors because they compete with the natural substrate for the active site (Figure 6.18a). In these cases, the inhibition is reversible. When the concentration of the competitive inhibitor is reduced, it detaches from the active site, and the enzyme is again active.

The enzyme succinate dehydrogenase is subject to competitive inhibition. This enzyme, found in all mitochondria, catalyzes the conversion of the compound succinate to fu-marate. A third molecule, oxaloacetate, is similar to succi-nate and can act as a competitive inhibitor of succinate de-hydrogenase by binding to its active site. Once bound to oxaloacetate, the enzyme can do nothing more with it—no reaction occurs. An enzyme molecule cannot bind a succi-

6.18 Reversible Inhibition (a) A competitive inhibitor binds temporarily to the active site of an enzyme. Succinate dehydrogenase, for example, is subject to competitive inhibition by oxaioacetate. (b) A noncompetitive inhibitor binds temporarily to the enzyme at a site away from the active site, but still blocks enzyme function.

nate molecule until the oxaioacetate molecule has moved out of its active site—which can occur if more substrate (succinate) molecules are added.

Some inhibitors that do not react with the active site are called noncompetitive inhibitors. Noncompetitive inhibitors bind to the enzyme at a site distinct from the active site. Their binding can cause a conformational change in the enzyme that alters the active site (Figure 6.18b). In this case, the active site may still bind substrate molecules, but the rate of product formation may be reduced. Noncompetitive inhibitors, like competitive inhibitors, can become unbound, so their effects are reversible.

Allosteric enzymes control their activity by changing their shape

The change in enzyme shape due to noncompetitive inhibitor binding is an example of allostery (allo-, "different"; -stery, "shape"). In that case, the binding of the inhibitor induces the protein to change its shape. More common are enzymes that

(a) Competitive inhibition

Substrate

(a) Competitive inhibition

Substrate

Functional Shape Enzyme

Inhibitor and substrate "compete;" only one can bind to the active site.

Enzyme

Inhibitor and substrate "compete;" only one can bind to the active site.

Enzyme

(b) Noncompetitive inhibition

The enzyme's function is disabled as long as the inhibitor remains bound.

Competitive inhibition of succinate dehydrogenase

Succinate (substrate)

Succinate (substrate)

Oxaioacetate (competitive inhibitor)

Fumarate

Fumarate

Catalyzed by succinate dehydrogenase

Oxaioacetate (competitive inhibitor)

Succinate dehydrogenase is subject to competitive inhibition by oxaloacetate, which resembles succinate enough to bind to the active site but cannot react.

Noncompetitive inhibition of threonine dehydratase

Substrate

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Essentials of Human Physiology

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