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Extended Lecture Outline |
Chapter 5: Enzymes And The Dynamics Of Proteins |
A. ENZYME FUNCTION
5.1 Enzymes work by lowering energy barriers.
a. In a chemical reaction, some bonds between atoms in the reactants are broken and new bonds are formed.
1. It takes energy to break bonds (Figure 5.1).
2. Reactants must cross an energy barrier to be converted into products.
3. In a chemical reaction, the reactants must have sufficient energy to temporarily form an activated complex or transition state, a fleeting combination of atoms that has more energy than either the reactants or products.
4. The activated complex changes into the products.
b. One way to increase the rate of a chemical reaction is to raise the temperature.
1. As temperature increases, molecules move faster and more of them will have enough energy to cross the barrier.
2. The factor by which the rate of the reaction increases when the temperature increases 10û C is its Q10.
c. Some organisms do depend on an increase in temperature to boost their metabolism.
1. Insects, turtles, and snakes will often lie in the sun to get warmer so they can move more quickly.
2. Metabolism always depends upon catalysts (e.g. enzymes).
d. Enzymes and other catalysts accelerate chemical reactions by reducing the energy barrier.
1. Enzymes lower the energy barrier that substrates must pass and increase their reaction rate.
2. The activity of an enzyme is often expressed by its turnover number. This is the number of substrate molecules transformed per second by each enzyme molecule (Table 5.1).
e. Enzymes are commonly named by adding -ase to the name of the substrate.
1. Protease is an enzyme that digests proteins. Sucrase splits the disaccharide sucrose into two simple sugars.
2. Some names describe the type of reactions that the enzyme catalyzes. Oxidases oxidize and ligases join (ligate) two molecules.
f. There are a few enzymes that were named before this standardized terminology was introduced. Pepsin and trypsin are two examples. They have retained their original names.
5.2 Chemical reactions come to an equilibrium if left undisturbed.
a. All chemical reactions proceed in at least one direction. Some chemical reactions are reversible and can proceed in either direction.
b. If a reversible reaction is allowed to proceed for a significant amount of time, it will reach a point of equilibrium, where the concentrations of the various materials involved in the reactions do not change.
c. Dynamic equilibrium is achieved when the forward and reverse reactions are still occurring at equilibrium, but they balance each other so the overall conditions do not change.
d. The equilibrium constant (Keq) of a chemical reaction is defined as the ratio [concentration of products]/[concentration of reactants] at equilibrium. The Keq predicts the direction of the reaction.
1. If the Keq is greater than 1, the reaction will proceed in the forward direction.
2. If the Keq is less than 1, the reaction will go in the reverse direction.
e. Catalysts, including enzymes, increase the rate of a reaction but they do not change the equilibrium because they increase the forward and reverse rates equally.
5.3 An enzyme's structure enables it to catalyze a specific reaction.
a. Each enzyme catalyzes a specific reaction, because it can only bind substrates whose shapes are complementary to its active site.
1. Amino acid side chains in the active site interact specifically with the substrate to lower the reactions energy barrier.
b. Lysozyme, discovered by Alexander Fleming, is an enzyme found in human tears, mucus, and egg white that cuts bonds in bacterial cell walls destroying the bacteria (Figure 5.3).
5.4 Michaelis—Menten kinetics shows that an enzyme has a limited number of active sites.
a. In 1913, Leonor Michaelis and Maude Menten developed a way to analyze enzyme-catalyzed reactions; their method is still in use today.
1. By using a constant amount of enzyme, they were able to study the kinetics of the reaction.
2. Kinetics is defined as how the rate or velocity of a reaction changes as the concentration of substrate, [S], changes (Figure 5.4).
3. When reaction rate is plotted against [S], a curve is produced that is characteristic of each enzyme. As [S] increases, the rate increases rapidly at first, but then levels off and doesn't increase further no matter how much additional substrate is added.
b. The reaction rate curve supports the view that each enzyme molecule has only one active site (or possibly a few sites) that interact with substrate molecules (Figure 5.5).
1. The point on the curve at which the reaction rate levels off at a maximum rate is called Vmax.
2. Since the number of active sites is limited, when all of them are occupied the enzymes are operating at maximum capacity. At this point, the enzymes are considered to be saturated.
c. Every enzyme can be characterized by a Michaelis constant, Km, which is defined as the substrate concentration at which the reaction rate is half of Vmax.
1. Km measures the tightness of binding, or affinity, between enzyme and substrate.
2. A high Km indicates loose binding (low affinity) between enzyme and substrate.
3. A low Km indicates tight binding (high affinity) between enzyme and substrate.
d. The Michaelis—Menten model pertains in general to many kinds of proteins, not just enzymes.
5.5 Temperature affects the rate of enzyme-catalyzed reactions.
a. Every enzyme has an optimal temperature at which it is most active.
1. Raising the environmental temperature increases enzyme activity simply by increasing the kinetic energy of molecules.
2. As the temperature is raised above the optimum, enzymes become denatured and their active sites can no longer bind substrates.
3. Each enzyme's optimal temperature represents a balance between increasing kinetic energy and protein denaturation.
b. Organisms have evolved with enzymes whose optimal temperatures are suited for the conditions in which the organisms live.
1. Biomolecules are shaped by evolution to function in a specific ecological niche.
2. Organisms that live at extreme temperatures have enzymes with amino acids that form more heat-stable bonds.
3. Organisms whose body temperatures vary widely have enzymes that function well over a broad temperature range.
4. The temperatures at which similar species of bacteria live are closely correlated with the temperatures at which their enzymes are denatured (Table 5.2).
5. Human body temperature is controlled close to 37û C, and that is the optimal temperature of most human enzymes.
5.6 Each enzyme has an optimal pH.
a. The pH of the solution around an enzyme also affects its structure and activity, because hydrogen ions interact with the polar groups on proteins and often take part in the reactions they catalyze.
1. Each enzyme has its own optimal pH.
2. The rate of enzyme-catalyzed reactions depends on pH.
3. Human blood has a pH of 7.4.
5.7 Some enzymes require cofactors and coenzymes to function.
a. The concentrations of ions such as sodium, potassium, magnesium, and chloride affect the rate of enzyme-catalyzed reactions because these ions interact with polar groups on an enzyme and subtly change their properties.
b. Several metal ions such as copper, zinc, nickel, and manganese act as enzyme cofactors.
1. Enzyme cofactors are bound to the active site of the enzyme and participate in the chemical reaction.
2. An enzyme that uses a cofactor requires a particular element; without it, the enzyme can't function.
c. Some enzymes also function only with coenzymes, which are certain small, non-protein organic molecules that are "helpers" needed to make an enzyme complete.
1. A coenzyme participates in a reaction by carrying in a small group of atoms, such as a methyl group (-CH3), or removing it from a substrate.
2. Many coenzymes are derived from vitamins, which are organic compounds that an organism requires in small amounts but cannot make for itself.
d. Prosthetic groups are similar to coenzymes but remain attached to the protein. Heme molecules bound to each polypeptide chain of hemoglobin are examples of these.
B. GENERAL PROTEIN FUNCTION.
5.8 Studies of enzyme inhibition help explain protein action and emphasize the importance of weak bonds between molecules.
a. Many enzymes are subject to competitive inhibition.
1. A competitive inhibitor is so similar in structure to the enzyme's normal substrate that it can occupy the active site and block entry by the substrate. In other words, it competes with the substrate for entrance into a limited number of active sites.
2. The competitor may be an alternate substrate or a molecule that is unable to enter into a chemical reaction as a substrate does.
b. Studies of enzyme kinetics make it clear that both the substrate and the inhibitor bind to amino-acid side chains in the active site through weak interactions.
c. Enzyme molecules and substrate molecules are involved in a dynamic equilibrium.
1. Enzyme molecules and substrate molecules combine to make a complex which can either go back to separate molecules or forward to produce the enzyme plus product.
2. If a competitive inhibitor is added, it forms weak bonds with the active site and there will be an equilibrium between inhibitors that are bound to the enzyme and those not bound. Competition will exist between substrate and inhibitor molecules.
d. Specific molecules enter a site based on their relative concentrations.
1. The molecule in higher concentration is more likely to bind to the site.
2. Inhibition can be overcome by raising the concentration of substrate because the molecules are continually binding and dissociating and the substrate molecules will tend to displace inhibitor molecules from the active sites.
e. Enzymes can also be affected by irreversible inhibitors.
1. Irreversible inhibitors form a covalent bond with part of the enzyme's active site, permanently destroying the enzyme's activity.
2. A historical example of irreversible inhibitors is the use of poison nerve gas (diisopropyl-phosphofluoridate) in World War I (Figure 5.6).
5.9 Hemoglobin illustrates cooperative effects between the subunits of a protein.
a. To investigate the way oxygen binds to hemoglobin and myoglobin, one can do an experiment very similar to the one used to study enzyme kinetics.
1. The experimental results are displayed as a saturation curve by plotting the fraction of protein molecules bound to O2 against the concentration of O2.
2. A saturation curve for myoglobin resembles the curve for the rate of an enzymatically catalyzed reaction with Michaelis—Menten kinetics (Figure 5.7).
3. Myoglobin has a single binding site for oxygen just as an enzyme has a single active site for binding its substrate.
4. More binding sites are occupied at increasing concentrations of oxygen until all the molecules are saturated.
b. The saturation curve for hemoglobin is sigmoid (S-shaped).
1. At relatively low concentrations of oxygen, only a small fraction of hemoglobin molecules are saturated.
2. Hemoglobin becomes saturated at a higher concentration of oxygen than is required to saturate myoglobin.
3. Myoglobin binds O2 more tightly than hemoglobin does.
c. The sigmoid shape of the saturation curve of hemoglobin is the result of a cooperative effect between the protein's subunits.
1. Each hemoglobin subunit can switch between two distinct conformations. These are known as the B and N conformations.
2. The B conformation is where O2 is bound to the heme.
3. The N conformation is where O2 is not bound to the heme.
4. Myoglobin does not show a cooperative effect because it has only one polypeptide chain and a single heme group that can bind O2.
5.10 The binding of proteins to other molecules is fundamental to biological processes.
a. Each enzyme interacts with another molecule, its substrate.
b. In general, biological processes occur because a protein interacts with a molecule called a ligand.
1. A ligand is most often a small molecule, but can occasionally be a macromolecule.
2. Oxygen is the principal ligand with which hemoglobin and myoglobin interact.
c. An enzyme-substrate interaction takes place in a specific place on the enzyme, its active site.
1. The binding site is the site on a protein where it binds to a ligand.
2. In hemoglobin and myoglobin, the oxygen-binding sites are at the heme groups.
d. The enzyme only binds a specific molecule with a structure that just fits its active site.
1. A protein only interacts with a ligand that specifically fits into its binding site.
e. Shape-specificity or stereospecificity is the name for interactions between molecules with shapes that fit together.
f. When molecules fit together perfectly in this way they are called complimentary.
g. The interaction between an enzyme and a substrate has the biological consequence of the substrate being converted into a product. Here are some other consequences that this interaction can have:
1. When a protein and its ligand interact, the protein simply holds the ligand and carries it for a while (e.g. as in the globins).
2. When a protein and its ligand interact, the protein transports the ligand across a cell membrane.
3. When a protein and its ligand interact, the protein prevents the ligand from having some other effect.
4. When a protein and its ligand interact, the ligand changes the protein's shape, thus either inhibiting the protein or activating it.
5.11 Many proteins, including hemoglobin, show allosteric effects.
a. The cooperativity exhibited by hemoglobin is one of a large class of actions called allosteric effects.
b. An allosteric protein is one that has two or more binding sites with quite different shapes that can bind ligands with correspondingly different shapes.
c. The function of hemoglobin is not just to bind oxygen, but also to release oxygen to the tissues that require it for metabolism.
d. Red blood cells contain about equimolar amounts of hemoglobin and BPG (2,3-bisphosphoglyceric acid), and each hemoglobin molecule has a site between the two b chains where BPG binds (Figure 5.8).
e. The place where BPG binds is a regulatory site that regulates the operation of hemoglobin. BPG acts as an allosteric effector, a ligand that changes the activity of an allosteric protein.
f. Carbon dioxide also affects the conformation of hemoglobin dramatically, although there is no binding site for CO2 such as the BPG binding site or the active site of an enzyme.
1. CO2 is produced abundantly in the metabolizing tissues of the body.
2. Hemoglobin does double duty by transporting both O2 and CO2, and by shifting from one conformation to the other, it does so very effectively.
5.12 A small change in protein structure can have profound functional consequences.
a. Even a small change in the basic structure of the protein can have enormous consequences.
1. Examples of this are the diseases anemia and sickle-cell anemia.
2. Most people have two copies of the normal gene symbolized by HbA and consequently they produce only normal hemoglobin, Hb A.
3. A small percentage of people have one HbA gene and one for hemoglobin S, HbS. In this case, they have both kinds of hemoglobin in their red blood cells and have essentially normal states of health. These people are carriers of the disease with the possibility of passing it on to their children.
4. A very small percentage of people have two copies of the HbS gene, so they produce only Hb S and become very ill. These people have sickle-cell anemia.
5. Under conditions such as reduced oxygen concentration, cells containing Hb S change into elongated or sickle-shaped cells. These cells clog small blood vessels and lead to many health problems (Figure 5.9).
5.13 The primary structure of a protein is a record of its evolutionary history.
a. Modern technology for sequencing proteins has quickly opened the door to comparative studies of proteins.
b. Evolution is shown by homologies between similar structures in different species. Proteins also show homology, sometimes very clearly.
1. Proteins with very similar sequences in different species are considered homologous, even though they may take on different forms and functions during the course of evolution.
2. Phylogenies based on proteins have generally confirmed phylogenies based on anatomy, and comparisons of many protein sequences from many species can refine phylogenies and clarify complex situations.
c. Homology takes on a different meaning when proteins with different functions but very similar sequences are found.
1. A phylogenetic tree of different globin chains shows that new proteins can become diversified to serve new functions (Figure 5.10).
2. Partial homologies exist where only some portion of two molecules show homology.
5.14 Computer searches help identify the function of an unknown protein on the basis of its structure.
a. Enormous databases of protein sequences are growing daily and are stored in central data banks, which scientists all over the world can tap into electronically via the internet.
1. Two prime examples of such databases are found at the National Institutes of Health in Bethesda, Maryland and the Japan International Protein Information Database (JIPID) in Tokyo, Japan (Figure 5.11).
2. Computers can compare the sequence of an otherwise unknown protein with all the sequences in the database and find those that show homologies to the newly sequenced protein.
3. Biologists are working in an ever-expanding background of molecular detail, and their work can advance rapidly by making use of everything that is already known about protein structure.
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