Virtually all reactions in the body are mediated by enzymes, which are protein catalysts that increase the rate of reactions without being changed in the overall process. Among the many biologic reactions that are energetically possible, enzymes selectively channel reactants (called substrates) into useful pathways. Enzymes thus direct all metabolic events. This chapter examines the nature of these catalytic molecules and their mechanism of action.
Each enzyme is assigned two names. The first is its short, recommended name, convenient for everyday use. The second is the more complete systematic name, which is used when an enzyme must be identified without ambiguity.
A. Recommended name
Most commonly used enzyme names have the suffix “-ase” attached to the substrate of the reaction (for example, glucosidase and urease), or to a description of the action performed (for example, lactate dehydrogenase and adenylyl cyclase). [Note: Some enzymes retain their original trivial names, which give no hint of the associated enzymic reaction, for example, trypsin and pepsin.]
B. Systematic name
In the systematic naming system, enzymes are divided into six major classes (Figure 5.1), each with numerous subgroups. For a given enzyme, the suffix -ase is attached to a fairly complete description of the chemical reaction catalyzed, including the names of all the substrates; for example, lactate:NAD+ oxidoreductase. [Note: Each enzyme is also assigned a classification number.] The systematic names are unambiguous and informative, but are frequently too cumbersome to be of general use.
Figure 5.1.The six major classes of enzymes with examples.
THF = tetrahydrofolate.
Potentially confusing enzyme nomenclature: synthetase (requires ATP), synthase (no ATP required); phosphatase (uses water to remove phosphoryl group), phosphorylase (uses Pi to break a bond and generate a phosphorylated product); dehydrogenase (NAD+/FAD is electron acceptor in redox reaction), oxidase (O2 is acceptor but oxygen atoms are not incorporated into substrate), oxygenase (one or both oxygens atoms are incorporated).
Properties of Enzymes
Enzymes are protein catalysts that increase the velocity of a chemical reaction, and are not consumed during the reaction. [Note: Some RNAs can act like enzymes, usually catalyzing the cleavage and synthesis of phosphodiester bonds. RNAs with catalytic activity are called ribozymes (see Shine-Dalgarno sequence), and are much less commonly encountered than protein catalysts.]
A. Active sites
Enzyme molecules contain a special pocket or cleft called the active site. The active site contains amino acid side chains that participate in substrate binding and catalysis (Figure 5.2). The substrate binds the enzyme, forming an enzyme–substrate (ES) complex. Binding is thought to cause a conformational change in the enzyme (induced fit) that allows catalysis. ES is converted to an enzyme–product (EP) complex that subsequently dissociates to enzyme and product.
Figure 5.2.Schematic representation of an enzyme with one active site binding a substrate molecule.
B. Catalytic efficiency
Enzyme-catalyzed reactions are highly efficient, proceeding from 103–108 times faster than uncatalyzed reactions. The number of molecules of substrate converted to product per enzyme molecule per second is called the turnover number, or kcat and typically is 102–104s?1.
Enzymes are highly specific, interacting with one or a few substrates and catalyzing only one type of chemical reaction. [Note: The set of enzymes made in a cell determines which metabolic pathways occur in that cell.]
Some enzymes require molecules other than proteins for enzymic activity. The term holoenzyme refers to the active enzyme with its nonprotein component, whereas the enzyme without its nonprotein moiety is termed an apoenzyme and is inactive. If the nonprotein moiety is a metal ion such as Zn2+ or Fe2+, it is called a cofactor. If it is a small organic molecule, it is termed a coenzyme. Coenzymes that only transiently associate with the enzyme are called cosubstrates. Cosubstrates dissociate from the enzyme in an altered state (NAD+ is an example, see Oxidation of glyceraldehyde 3-phosphate). If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group (FAD is an example, see Coenzymes). Coenzymes frequently are derived from vitamins. For example, NAD+ contains niacin and FAD contains riboflavin (see Chapter 28).
Enzyme activity can be regulated, that is, increased or decreased, so that the rate of product formation responds to cellular need.
F. Location within the cell
Many enzymes are localized in specific organelles within the cell (Figure 5.3). Such compartmentalization serves to isolate the reaction substrate or product from other competing reactions. This provides a favorable environment for the reaction, and organizes the thousands of enzymes present in the cell into purposeful pathways.
Figure 5.3.The intracellular location of some important biochemical pathways.
How Enzymes Work
The mechanism of enzyme action can be viewed from two different perspectives. The first treats catalysis in terms of energy changes that occur during the reaction, that is, enzymes provide an alternate, energetically favorable reaction pathway different from the uncatalyzed reaction. The second perspective describes how the active site chemically facilitates catalysis.
A. Energy changes occurring during the reaction
Virtually all chemical reactions have an energy barrier separating the reactants and the products. This barrier, called the free energy of activation, is the energy difference between that of the reactants and a high-energy intermediate that occurs during the formation of product. For example, Figure 5.4 shows the changes in energy during the conversion of a molecule of reactant A to product B as it proceeds through the transition state (high-energy intermediate), T*:
Figure 5.4.Effect of an enzyme on the activation energy of a reaction.
1. Free energy of activation
The peak of energy in Figure 5.4 is the difference in free energy between the reactant and T*, where the high-energy intermediate is formed during the conversion of reactant to product. Because of the high free energy of activation, the rates of uncatalyzed chemical reactions are often slow.
2. Rate of reaction
For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state. In the absence of an enzyme, only a small proportion of a population of molecules may possess enough energy to achieve the transition state between reactant and product. The rate of reaction is determined by the number of such energized molecules. In general, the lower the free energy of activation, the more molecules have sufficient energy to pass through the transition state, and, thus, the faster the rate of the reaction.
3. Alternate reaction pathway
An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower free energy of activation (Figure 5.4). The enzyme does not change the free energies of the reactants or products and, therefore, does not change the equilibrium of the reaction (see Standard free energy change, ?Go). It does, however, accelerate the rate with which equilibrium is reached.
B. Chemistry of the active site
The active site is not a passive receptacle for binding the substrate, but rather is a complex molecular machine employing a diversity of chemical mechanisms to facilitate the conversion of substrate to product. A number of factors are responsible for the catalytic efficiency of enzymes, including the following:
1. Transition-state stabilization
The active site often acts as a flexible molecular template that binds the substrate and initiates its conversion to the transition state, a structure in which the bonds are not like those in the substrate or the product (see T* at the top of the curve in Figure 5.4). By stabilizing the transition state, the enzyme greatly increases the concentration of the reactive intermediate that can be converted to product and, thus, accelerates the reaction.
2. Other mechanisms
The active site can provide catalytic groups that enhance the probability that the transition state is formed. In some enzymes, these groups can participate in general acid-base catalysis in which amino acid residues provide or accept protons. In other enzymes, catalysis may involve the transient formation of a covalent ES complex. [Note: The mechanism of action of chymotrypsin, an enzyme of protein digestion in the intestine, includes general base, general acid, and covalent catalysis. A histidine at the active site of the enzyme gains (general base) and loses (general acid) protons, mediated by the pK of histidine in proteins being close to physiologic pH. Serine at the active site forms a covalent link with the substrate.]
3. Visualization of the transition state
The enzyme-catalyzed conversion of substrate to product can be visualized as being similar to removing a sweater from an uncooperative infant (Figure 5.5). The process has a high energy of activation because the only reasonable strategy for removing the garment (short of ripping it off) requires that the random flailing of the baby results in both arms being fully extended over the head—an unlikely posture.
However, we can envision a parent acting as an enzyme, first coming in contact with the baby (forming ES), then guiding the baby’s arms into an extended, vertical position, analogous to the ES transition state. This posture (conformation) of the baby facilitates the removal of the sweater, forming the disrobed baby, which here represents product. [Note: The substrate bound to the enzyme (ES) is at a slightly lower energy than unbound substrate (S) and explains the small “dip” in the curve at ES.]
Figure 5.5. Schematic representation of energy changes accompanying formation of an enzyme–substrate complex and subsequent formation of a transition state.
Factors Affecting Reaction Velocity
Enzymes can be isolated from cells, and their properties studied in a test tube (that is, in vitro). Different enzymes show different responses to changes in substrate concentration, temperature, and pH. This section describes factors that influence the reaction velocity of enzymes. Enzymic responses to these factors give us valuable clues as to how enzymes function in living cells (that is, in vivo).
A. Substrate concentration
1. Maximal velocity
The rate or velocity of a reaction (v) is the number of substrate molecules converted to product per unit time; velocity is usually expressed as ?mol of product formed per minute. The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached (Figure 5.6). The leveling off of the reaction rate at high substrate concentrations reflects the saturation with substrate of all available binding sites on the enzyme molecules present.
Figure 5.6.Effect of substrate concentration on reaction velocity.
2. Hyperbolic shape of the enzyme kinetics curve
Most enzymes show Michaelis-Menten kinetics (see Michaelis-Menten Equation), in which the plot of initial reaction velocity (vo) against substrate concentration ([S]), is hyperbolic (similar in shape to that of the oxygen-dissociation curve of myoglobin, see Myoglobin (Mb)). In contrast, allosteric enzymes do not follow Michaelis-Menton kinetics and show a sigmoidal curve (see Homotropic effectors) that is similar in shape to the oxygen dissociation curve of hemoglobin (see Hemoglobin (Hb)).
1. Increase of velocity with temperature
The reaction velocity increases with temperature until a peak velocity is reached (Figure 5.7). This increase is the result of the increased number of molecules having sufficient energy to pass over the energy barrier and form the products of the reaction.
Figure 5.7.Effect of temperature on an enzyme-catalyzed reaction.
2. Decrease of velocity with higher temperature
Further elevation of the temperature results in a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme (see Figure 5.7).
The optimum temperature for most human enzymes is between 35 and 40°C. Human enzymes start to denature at temperatures above 40°C, but thermophilic bacteria found in the hot springs have optimum temperatures of 70°C.
1. Effect of pH on the ionization of the active site
The concentration of H+ affects reaction velocity in several ways. First, the catalytic process usually requires that the enzyme and substrate have specific chemical groups in either an ionized or un-ionized state in order to interact. For example, catalytic activity may require that an amino group of the enzyme be in the protonated form (–NH3+). At alkaline pH, this group is deprotonated, and the rate of the reaction, therefore, declines.
2. Effect of pH on enzyme denaturation
Extremes of pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid side chains.
3. The pH optimum varies for different enzymes
The pH at which maximal enzyme activity is achieved is different for different enzymes, and often reflects the [H+] at which the enzyme functions in the body. For example, pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other enzymes, designed to work at neutral pH, are denatured by such an acidic environment (Figure 5.8).
Figure 5.8.Effect of pH on enzyme-catalyzed reactions.