Enzyme

I OVERVIEW
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 biological reactions that are energetically possible, enzymes selectively channel reactants (called substrates) into useful pathways. Enzymes thus direct all metabolic events.
II. NOMENCLATURE
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, urease, sucrase), 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
The International Union of Biochemistry and Molecular Biology (IUBMB) developed a system of nomenclature in which enzymes are divided into six major classes (Figure 5.1), each with numerous subgroups. The suffix -ase is attached to a fairly complete description of the chemical reaction catalyzed, including the names of all the substrates; for example D-glyceraldehyde 3-phosphate:NAD oxidoreductase. The IUBMB names are unambiguous and informative, but are frequently too cumbersome to be of general use.
Enzyme Commission Number (EC number) The Enzyme Commission number (EC number) is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. As a system of enzyme nomenclature, every EC number is associated with a recommended name of enzyme. Every enzyme code consists of the letters "EC" followed by four numbers separated by periods. Those numbers represent a progressively finer classification of the enzyme. For example, the tripeptide aminopeptidases have the code "EC 3.4.11.4", whose components indicate the following groups of enzymes: ? EC 3 enzymes are hydrolases (enzymes that use water to break up some other molecule) ? EC 3.4 are hydrolases that act on peptide bonds ? EC 3.4.11 are those hydrolases that cleave off the amino-terminal amino acid from a polypeptide ? EC 3.4.11.4 are those that cleave off the amino-terminal end from a tripeptide
III. PROPERTIES OF ENZYMES
Enzymes are protein catalysts that increase the velocity of a chemical reaction, and are not consumed during the reaction they catalyze. [Note: Some types of RNA can act like enzymes, usually catalyzing the cleavage and synthesis of phosphodiester bonds. RNAs with catalytic activity are called ribozymes, 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 create a three-dimensional surface complementary to the substrate (Figure 5.2). The active site binds the substrate, forming an enzyme–substrate (ES) complex. ES is converted to an enzyme–product (EP) complex that subsequently dissociates to enzyme and product.
B. Catalytic efficiency
Most enzyme-catalyzed reactions are highly efficient, proceeding from 103 to 108 times faster than uncatalyzed reactions. Typically, each enzyme molecule is capable of transforming 100 to 1000 substrate molecules into product each second. The number of molecules of substrate converted to product per enzyme molecule per second is called the turnover number, or kcat.
C. Specificity
Enzymes are highly specific, interacting with one or a few substrates and catalyzing only one type of chemical reaction.
Note: Because enzymes are extremely selective for their substrates, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.
D. Holoenzymes
Some enzymes require molecules other than proteins for enzymic activity. 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+ and coenzyme A are examples,). If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group (FAD is anexample). Coenzymes frequently are derived from vitamins. For example, NAD+ contains niacin, coenzyme A contains pantothenic acid, and FAD contains riboflavin.
E. Regulation
Enzyme activity can be regulated, that is, enzymes can be activated or inhibited, so that the rate of product formation responds to the needs of the cell.
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.
IV. 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*:
A ? T* ?B
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. 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 in a geometric structure resembling the activated transition state of the molecule. By stabilizing the substrate in its 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 enzyme-substrate complex. 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 looses (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. 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 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.]
V. FACTORS AFFECTING REACTION VELOCITY
Enzymes can be isolated from cells, and their properties studied in a test tube (in vitro). Different enzymes show different responses to changes in substrate concentration, temperature, and pH. Enzymic responses to these factors give us valuable clues as to how enzymes function in living cells.
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 enzymecatalyzed
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.
2. Hyperbolic shape of the enzyme kinetics curve: Most enzymes show Michaelis-Menten
kinetics, 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). In
contrast, allosteric enzymes frequently show a sigmoidal curve that is similar in shape to the
oxygen-dissociation curve of hemoglobin.
B. Temperature
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.
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).
Note: The optimum temperature for most human
enzymes is between 35 and 40°C. Human enzymes start to denature at temperatures
above 40°C, but thermophil bacteria found in the hot springs have optimum
temperatures of 70 °C.
C. pH
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 unionized 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).