Chemistry 20 Chapters 15 Enzymes Enzymes: as a catalyst, an enzyme increases the rate of a reaction by changing the way a reaction takes place, but is itself not changed at the end of the reaction. An unanalyzed reaction is a cell may take place eventually, but not at a rate fast enough for survival. For example, the hydrolysis of proteins in our diet would eventually occur without a catalyst, but not fast enough to meet the body s requirements for amino acids. The chemical reactions in our cells must occur at incredibly fast rates under the mild conditions pf ph 7.4 and a body temperature of 37 C. To do this, biological catalysts known as enzymes catalyze nearly all the chemical reactions that take place in the body. As catalysts, enzymes lower the activation energy for the reaction (activation energy is the minimum energy necessary to cause a chemical reaction to occur). As a result, less energy is required to convert reactant molecules to products, which allows more reacting molecules to form product. E act E act Note: The vast majority of all known enzymes are globular proteins. However, proteins are not the only biological catalysts. Note: Most of enzymes are extremely specific (catalyze specific reactions). Some enzymes are localized according to the need for specific reaction (for example, digestive enzymes, which catalyze the hydrolysis of proteins, are located in the secretions of the stomach and pancreas). Names of enzymes: the names of enzymes describe the compound or the reaction that is catalyzed. The actual names of enzymes are derived by replacing the end of the name of the reaction or reacting compound with the suffix -ase. For example, an oxidase catalyzes an oxidation reaction, and a dehydrogenase removes hydrogen atoms. The compound amylose is hydrolyzed by the enzyme amylase, and a lipid is hydrolyzed by a lipase. Some early known enzymes use names that end in the suffix -in, such as papain found in papaya, rennin found in milk, and pepsin and trypsin, enzymes that catalyze the hydrolysis of proteins. There are six classes of enzymes:
Enzyme action: Nearly enzymes are globular proteins. Each has a unique three-dimensional shape that recognizes and binds a small group of reacting molecules, which are called substrates. The tertiary structure of an enzyme plays an important role in how that enzyme catalyzes reactions. Active site: in a catalyzed reaction, an enzyme must first bind to a substrate in away that favors catalysis. A typical enzyme is much larger that its substrate. However, within its large tertiary structure, there is a region called the active site where the enzyme binds a substrate or substrates and catalyzes the reaction. This active site is often a small pocket that closely fits the structure of the substrate. Within the active site, the side chains of amino acids bind the substrate with hydrogen bonds, salt bridges, or hydrophobic attractions. The active site of a particular enzyme fits the shape of only a few types of substrates, which makes enzymes very specific about the type of substrate they bind. Enzyme catalyzed reaction: the proper alignment of a substrate within the active site forms an enzyme-substrate (ES) complex. This combination of enzyme and substrate provides an alternative pathway for the reaction that has a lower activation energy. Within the active site, the amino acid side chains take part in catalyzing the chemical reaction. As soon as the catalyzed reaction is complete, the products are quickly released from the enzyme so it can bind to a new substrate molecule. We can write the catalyzed reaction of an enzyme (E) with a substrate (S) to form product (P) as follows: E (Enzyme) + S (Substrate) ES (Complex) E (Enzyme) + P (Product) Let s consider the hydrolysis of sucrose by sucrase (enzyme). When sucrose binds to the active site of sucrase, the glycosidic bond of sucrose is placed into a geometry favorable for reaction. The amino acid side chains catalyze the cleavage of the sucrose to give the products glucose and fructose. Because the structures of the products are no longer attracted to the active site, they are releases and the sucrase binds another sucrose substrate. Sucrase (E) + Sucrose (S) Sucrase-Sucrose complex Sucrase (E) + glucose (P2) + Fructose (P2) There are two models for formation of ES Complex: Dr. Behrang Madani Chemistry 20 Mt SAC
1. Lock-and-Key model: in this theory, the active site is described as having a rigid, nonflexible shape. Thus only those substrates with shapes that fit exactly into the active site are able to bind with enzyme. The shape of the active site is analogous to a lock, and the proper substrate is the key that fits into the lock. Problems: This model is too restrictive. Enzyme molecules are in a dynamic state, not a static one. There are constant motions within them, so that active site has some flexibility. Also, if the fit between substrate and active site is prefect, there would be no reason for the reaction to occur, as the enzyme-substrate complex would be too stable! From X-ray diffraction, we know that the size and shape of the active site cavity change when the substrate enters. 2. Induced-Fit model: certain enzymes have a broader range of activity that the lock and key model allows. In the induced-fit model, enzyme structure is flexible, not rigid. There is an interaction between both the enzyme and substrate. The active site adjusts to fit the shape of the substrate more closely. At the same time the substrate adjusts its shape to better adapt to the geometry of the active site. As a result, the reacting section of the substrate becomes aligned exactly with the groups in the active site that catalyze the reaction. A different substrate could not induce these structural changes and no catalysis would occur. Factors affecting enzyme activity: the activity of an enzyme describes how fast an enzyme catalyzed the reaction that converts a substrate to product. This activity is strongly affected by reaction conditions, which include the temperature, ph, concentration of the substrate or enzyme and the presence of inhibitors. 1. Temperature: enzymes are very sensitive to temperature. At low temperature, most enzymes show little activity because there is not a sufficient amount of energy for the catalyzed reaction to take place. At higher temperatures, enzyme activity increases as reacting
molecules move faster to cause more collisions with enzymes. Enzymes are most active at optimum temperature, which is 37 C or body temperature for most enzymes. At temperature above 50 C, the tertiary structure and thus the shape of most proteins is destroyed, which causes a loss in enzyme activity. For this reason, equipment in hospitals and laboratories is sterilized in autoclaves where the high temperatures denature the enzyme in harmful bacteria. 2. ph: enzymes are most active at their optimum ph, the ph that maintains the proper tertiary structure of the protein. A ph value above or below the optimum ph causes a change in he three-dimensional structure of the enzyme that disrupts the active site. As a result the enzyme cannot bind substrate properly and no reaction occurs. Enzymes in most cells have optimum ph values at physiological ph values around 7.4. However, enzymes in the stomach have a low optimum ph because they hydrolyze proteins at the acidic ph in the stomach. For example, pepsin, a digestive enzyme in the stomach, has an optimum ph of 2. Between meals, the ph in the stomach is 4 or 5 and pepsin shows little or no digestive activity. When food enters the stomach, the secretion of HCl lowers the ph to about 2, which actives pepsin. If small changes in ph are corrected, an enzyme can regain its structure and activity. However, large variations from optimum ph permanently destroy the structure of the enzyme. 3. Substrate and enzyme concentration: in any catalyzed reaction, the substrate must first bind with the enzyme to form the substrate-enzyme complex. When enzyme concentration increases, the rate of catalyzed reaction increases because we produce more substrate-enzyme complex. When enzyme concentration is kept constant, increasing the substrate concentration increases the rate of the catalyzed reaction as long as there are more enzyme molecules present than substrate molecules. At some point an increase in substrate concentration saturates the enzyme. With all the available enzyme molecules bonded to substrate, the rate of the catalyzed reaction reaches its maximum. Adding more substrate molecules cannot increase the rate further. Dr. Behrang Madani Chemistry 20 Mt SAC
Maximum activity 4. Enzyme inhibition: many kinds of molecules called inhibitors cause enzymes to lose catalytic activity. Although inhibitors act differently, they are all prevent the active site from binding with a substrate. Inhibition can be competitive or noncompetitive. Competitive inhibitor: a competitive inhibitor has a structure that is so similar to the substrate it competes for the active site on the enzyme. As long as the inhibitor occupies the active site, the substrate cannot bind to the enzyme and no reaction takes place. As long as the concentration of the inhibitor is substantial, there is a loss of enzyme activity. However, increasing the substrate concentration displaces more of the inhibitor molecules. As more enzyme molecules bind to substrate (ES), enzyme activity is regained. Noncompetitive inhibitor: the structure of a noncompetitive inhibitor does not resemble the substrate and does not compete for the active site. Instead, a noncompetitive inhibitor binds to a site on the enzyme that is not the active site. When the noncompetitive inhibitor is bonded to the enzyme, the shape of the enzyme is distorted. Inhibition occurs because the substrate cannot fit in the active site, or it does not fit properly. Without the proper alignment of substrate with the amino acid side groups, no catalysis can take place. Because a noncompetitive inhibitor is not competing for the active site, the addition of more substrate does not reverse this type of inhibition. Example of noncompetitive inhibitors are the heavy metal ions Pb 2+, Ag +, and Hg 2+ that bond with amino acid side groups such as COO -, or OH. Catalytic activity is restored when chemical reagents remove the inhibitors. Antibiotics produced by bacteria, mold, or yeast are inhibitors used to stop bacterial growth. For example, penicillin inhibits an enzyme needed for the formation of cell walls in bacteria, but not human cell membranes. With an incomplete wall, bacteria cannot survive, and the infection is stopped. However, some bacteria are resistant to penicillin because they produce penicillinase, an enzyme that breaks down penicillin. Over the years, derivatives of penicillin to which bacteria have not yet become resistant have been produced.
Inhibitor Site Enzyme cofactors: enzymes are known as simple enzyme (apoenzyme) when their function forms consist only of proteins with tertiary structure. However, many enzymes require small molecules or metal ions called cofactors to catalyze reactions properly. When the cofactor is a small organic molecule, it is known as a coenzyme. If an enzyme requires a cofactor, neither the protein structure nor the cofactor alone has catalytic activity.
protein Simple enzyme protein Metal ion Enzyme + Cofactor protein Organic molecules (coenzyme) Enzyme + Cofactor Metal ions: many enzymes must contain a metal ion to carry out their catalytic activity. The metal ions are bonded to one or more of the amino acid side chains. The metal ions from the minerals that we obtain from foods in our diet have various functions in catalysis. Ions such as Fe 2+ and Cu 2+ are used by oxidases because they lose or gain electrons in oxidation or reduction reactions. Other metals ions such as Zn 2+ stabilize the amino acid side chains during hydrolysis reactions. Vitamins and coenzymes: vitamins are organic molecules that are essential for normal health and growth. They are required in trace amounts and must be obtained from the diet because they are not synthesized in the body. Vitamins are classified into two groups by solubility: water-soluble and fat-soluble. Water-soluble vitamins: have polar groups such as OH and COOH, which make them soluble in the aqueous environment of the cells. Most water-soluble enzymes are not stored in the body and excess amounts are eliminated in the urine each day. Therefore, the watersoluble vitamins must be in the foods of our daily diets. Because many water-soluble vitamins are easily destroyed by heat, oxygen, and ultraviolet light, care must be taken in food preparation, processing, and storage. The water-soluble vitamins are required by many enzymes as cofactors to carry out certain aspects of catalytic action. The coenzymes do not remain bonded to a particular enzyme, but are used over and over again by different enzymes to facilitate an enzyme-catalyzed reaction. Thus, only small amounts of coenzymes are required in the cells.
Fat-soluble vitamins: are nonpolar compounds, which are soluble in the fat (lipid) components of the body such as fat deposits and cell membranes. The fat-soluble vitamins A, D, E, and K are not involved as coenzymes, but they are important in processes such as vision, formation of bone, protection from oxidation, and proper blood clotting. Because the fat-soluble vitamins are stored in the body and not eliminated, it is possible to take too much, which could be toxic. Enzyme regulation: 1. Feedback control: Enzymes are often regulated by environmental conditions. Feedback control is an enzyme regulation process in which formation of a product inhibits an earlier reaction in the sequence. The reaction product of one enzyme may control the activity of another (in the following reaction, D, may inhibit the activity of enzyme E 1 (by competitive are noncompetitive, or some other type of inhibition)). feedback inhibition E A 1 E B 2 E C 3 D 2. Proenzymes (Zymogens): some enzymes are manufactured by the body in an inactive form. To take them active, a small part of their polypeptide chain must be removed. These inactive forms of enzymes are called proenzymes or zymogens. After excess polypeptide chain is removed, the enzyme becomes active. For example, trypsin (an important catalyst for the digestion of the proteins) is manufactured as the inactive molecule trypsinogen (a zymogen). When a fragment containing six amino acid residues is removed from the N- terminal end of trypsinogen, the molecule becomes a fully active trypsin molecule. 3. Allosterism: sometimes regulation take place by means of an event that occurs site other than the active site but that eventually affects the active site. This type of interaction is called allosterism, and any enzyme regulated by this mechanism is called an allosteric enzyme. If a substance binds noncovalently and reversibly to a site other the active site, it may affect the enzyme in either of two ways: 1. It may inhibit enzyme action (negative modulation) or 2. It may stimulate enzyme action (positive modulation).
4. Protein modification: the activity of an enzyme may also be controlled by protein modification. The modification is usually a change in the primary structure, typically by addition of a functional group covalently bound to the apoenzyme. For example, the enzyme pyruvate kinase (PK) from the liver is inactive when it is phosphorylated. When the activity of PK is not needed, it is phosphorylated (to PKP) by a protein kinase using ATP as substrate as well as a source of energy. When the system wants to turn on PK activity, the phosphate group (Pi) is removed by another enzyme (phosphatase), which renders PK active. active PK ATP AD P kinase phosphatase PKP inactive P i H 2 O 5. Isoenzymes: another type of regulation of enzyme activity occurs when the same enzyme appears in different forms in different tissues. These different forms of the same enzyme are called isoenzymes. Isoenzymes perform the same function but have different combinations of subunits and thus different quaternary structure. Enzymes in medicine: most enzymes are confined within the cells of the body. However, small amounts of them can also be found in body fluids such as blood, and urine. The level of enzyme activity in these fluids can easily be monitored. This information can prove extremely useful: Abnormal activity (either high or low) of particular enzymes in various body fluids signals either the onset of certain diseases or their progression.