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Outline. 19.1 Catalysis by Enzymes 19.2 Enzyme Cofactors 19.3 Enzyme Classification 19.4 How Enzymes Work 19.5 Effect of Concentration on Enzyme Activity 19.6 Effect of Temperature and pH on Enzyme Activity 19.7 Enzyme Regulation: Feedback and Allosteric Control
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Outline 19.1 Catalysis by Enzymes 19.2 Enzyme Cofactors 19.3 Enzyme Classification 19.4 How Enzymes Work 19.5 Effect of Concentration on Enzyme Activity 19.6 Effect of Temperature and pH on Enzyme Activity 19.7 Enzyme Regulation: Feedback and Allosteric Control 19.8 Enzyme Regulation: Inhibition 19.9 Enzyme Regulation: Covalent Modification and Genetic Control 19.10 Vitamins and Minerals
Goals 1. What are enzymes? Be able to describe the chemical nature of enzymes and their function in biochemical reactions. 2. How do enzymes work, and why are they so specific? Be able to provide an overview of what happens as one or more substrates and an enzyme come together so that the catalyzed reaction can occur, and be able to list the properties of enzymes that make their specificity possible. 3. What effects do temperature, pH, enzyme concentration, and substrate concentration have on enzyme activity? Be able to describe the changes in enzyme activity that result when temperature, pH, enzyme concentration, or substrate concentration change. • How is enzyme activity regulated? Be able to define and identify feedback control, allosteric control, reversible and irreversible inhibition, inhibition by covalent modification, and genetic control of enzymes. • What are vitamins and minerals? Be able to describe the two major classes of vitamins, the reasons vitamins are necessary in our diets, and the general results of excesses or deficiencies. Be able to identify essential minerals, explain why minerals are necessary in the diet, and explain the results of deficiencies.
19.1 Catalysis by Enzymes • An enzyme is a protein or other molecule that acts as a catalyst for a biological reaction. • Enzymes, with few exceptions, are water-soluble globular proteins. • An active site is a pocket in an enzyme with the specific shape and chemical makeup necessary to bind a substrate. • A substrate is a reactant in an enzyme-catalyzed reaction. • Specificity of the enzyme is the limitation of the activity of an enzyme to a specific substrate, specific reaction, or specific type of reaction.
19.1 Catalysis by Enzymes • Catalase is almost completely specific for one reaction: the decomposition of hydrogen peroxide. • Thrombin catalyzes hydrolysis of a peptide bond following an arginine, and primarily acts on fibrinogen, a protein essential to blood clotting. • Carboxypeptidase A removes many different C-terminal amino acid residues from protein chains during digestion. • Papain catalyzes the hydrolysis of peptide bonds in many locations.
19.1 Catalysis by Enzymes • Enzymes are also specific with respect to stereochemistry. • If a substrate is chiral, an enzyme usually catalyzes the reaction of only one of the pair of enantiomers. • Only one enantiomer fits the active site in such a way that the reaction can occur.
19.1 Catalysis by Enzymes • The catalytic activity of an enzyme is measured by its turnover number, the maximum number of substrate molecules acted upon by one molecule of enzyme per unit time. • Turnover numbers range from 10 to 10,000,000 molecules per second. • Catalase is one of the fastest; it can turn over 10 million molecules per second, which is the fastest reaction rate attainable, because it is the rate at which molecules collide.
19.2 Enzyme Cofactors • An enzyme may require a metal ion, a coenzyme, or both. • Cofactors can be tightly held or loosely bound, so that they can enter and leave the active site. • A cofactor is a nonprotein part of an enzyme that is essential to the enzyme’s catalytic activity (e.g., a metal ion or a coenzyme). • A coenzyme is an organic molecule that acts as an enzyme cofactor.
19.2 Enzyme Cofactors • By combining with cofactors, enzymes acquire chemically reactive groups not available in side chains. • The requirement that many enzymes have for metal ion cofactors explains our dietary need for trace minerals. • Vitamins are also a necessity for humans; we cannot synthesize them, yet they are critical building blocks for coenzymes.
19.2 Enzyme Cofactors • By combining with cofactors, enzymes acquire chemically reactive groups not available in side chains. • The requirement for metal ion cofactors explains our dietary need for trace minerals. The ions form coordinate covalent bonds with nitrogen or oxygen. • Vitamins are also a necessity for humans; we cannot synthesize them, yet they are critical building blocks for coenzymes.
19.3 Enzyme Classification • Oxidoreductases catalyze oxidation–reduction reactions of substrate molecules, most commonly addition or removal of oxygen or hydrogen. Because oxidation and reduction must occur together, these enzymes require coenzymes that are reduced or oxidized as the substrate is oxidized or reduced.
19.3 Enzyme Classification • Transferases catalyze transfer of a group from one molecule to another. • Transaminases transfer an amino group between substrates. • Kinases transfer a phosphate group from adenosine triphosphate (ATP) to produce adenosine diphosphate (ADP) and a phosphorylated product.
19.3 Enzyme Classification • Hydrolases catalyze the hydrolysis of substrates, the breaking of bonds with addition of water. These enzymes are particularly important during digestion, and provide amino acids for protein synthesis and glucose for use in energy generating pathways.
19.3 Enzyme Classification • Isomerases catalyze the isomerization (rearrangement of atoms) of a substrate in reactions that have but one substrate and one product. In some metabolic pathways a molecule must be rearranged—isomerized—for the next step of the pathway to occur.
19.3 Enzyme Classification • Lyases (from the Greek lein, meaning “to break”) catalyze the addition of a molecule such as H2O, CO2 or NH3 to a double bond or the reverse reaction in which a molecule is eliminated to create a double bond.
19.3 Enzyme Classification • Ligases (from the Latin ligare, meaning “to tie together”) catalyze the bonding together of two substrate molecules. Because such reactions are generally not favorable, they require the simultaneous release of energy by a hydrolysis reaction, usually by the conversion of ATP to ADP. Ligases are involved in synthesis of biological polymers such as proteins and DNA.
19.4 How Enzymes Work • The explanation for enzyme specificity is found in the active site. Exactly the right environment for the reaction is provided within the active site. • Two models represent the interaction between substrates and enzymes: • In the lock-and-key model, the substrate is described as fitting into the active site as a key fits into a lock. • In the induced-fit model, the enzyme has a flexible active site that changes shape.
19.4 How Enzymes Work • Enzyme-catalyzed reactions begin with migration of the substrate into the active site to form an enzyme–substrate complex. • Enzymes act as catalysts because of their ability to: • Bring substrate(s) and catalytic sites together (proximity effect). • Hold substrate(s) at the exact distance and in the exact orientation necessary for reaction (orientation effect). • Provide acidic, basic, or other types of groups required for catalysis (catalytic effect). • Lower the energy barrier by inducing strain in bonds in the substrate molecule (energy effect).
19.5 Effect of Concentration on Enzyme Activity Substrate Concentration • If the substrate concentration is low, not all the enzyme molecules are in use. The rate increases with the concentration of substrate as more enzyme molecules are put to work. • As the substrate concentration continues to increase, the increase in the rate levels off as more and more active sites are occupied.
19.5 Effect of Concentration on Enzyme Activity Substrate Concentration • Once the enzyme is saturated, increasing substrate concentration has no effect. • The rate when the enzyme is saturated is determined by the efficiency of the enzyme, the pH, and the temperature. • Enzyme and substrate molecules moving at random in solution can collide with each other at a rate of about 108 collisions per mole per liter per second. • A few enzymes actually operate with close to this efficiency.
19.5 Effect of Concentration on Enzyme Activity Enzyme Concentration • It is possible for the concentration of an active enzyme to vary according to metabolic needs. So long as the concentration of substrate does not become a limitation, the reaction rate varies directly with the enzyme concentration.
19.6 Effect of Temperature and pH on Enzyme Activity Effect of Temperature on Enzyme Activity • Rates of enzyme-catalyzed reactions do not increase continuously with rising temperature. • Rates reach a maximum and then decrease. Enzymes denature when non-covalent attractions between protein side chains are disrupted, destroying the active site.
19.6 Effect of Temperature and pH on Enzyme Activity Effect of pH on Enzyme Activity • The catalytic activity of many enzymes depends on pH and usually has a well-defined optimum point at the normal, buffered pH of the enzyme’s environment. • Most enzymes have their maximum activity between the pH values of 5 to 9. Eventually, extremes of pH will denature a protein.
19.6 Effect of Temperature and pH on Enzyme Activity Extremozymes: Enzymes from the Edge • Most mammalian enzymes display optimum activity around 40 °C near pH 7.0 at 1 atmosphere of pressure. • Extremozymes are enzymes from extremophiles, organisms that live in conditions hostile to mammalian cells. • Commercially-useful enzymes have been developed from bacteria that have optimum growth temperatures as high as 106 °C and as low as 4 °C, and that grow in pH as low as 0.7 and as high as 10. • Enzymes from thermophiles (heat lovers) are used to break down starch and cellulose, and Taq polymerase is used in forensics. • Enzymes from cold-environment microorganisms (psychrophiles) are used in products such as cold-water-wash laundry detergents. • Thermophiles synthesize special proteins called chaperonins, which recognize and refold heat-denatured proteins. In addition, proteins from thermophiles have tightly folded, highly nonpolar cores, and ionic surfaces. • Psychrophiles have more polar, flexible proteins than thermophiles. This structure is necessary to maintain activity at low temperatures. • Thermophilic enzymes are also used in oil drilling.
19.6 Effect of Temperature and pH on Enzyme Activity Enzymes in Medical Diagnosis • Measurement of blood levels of enzymes is a valuable diagnostic tool. Higher-than-normal activities indicate the following conditions: • Aspartate transaminase (AST) Damage to heart or liver • Alanine transaminase (ALT) Damage to heart or liver • Lactate dehydrogenase (LH) Damage to heart, liver, or red blood cells • Alkaline phosphatase (ALP) Damage to bone and liver cells • Glutamyl transferase (GGT) Damage to liver cells; alcoholism • Activity is measured in international units. Results are reported in units per liter (U/L). • Among the most useful enzyme assays are those done to diagnose heart attacks.
19.7 Enzyme Regulation: Feedback and Allosteric Control • A variety of strategies are utilized to adjust the rates of enzyme-catalyzed reactions. • Any process that initiates or increases the action of an enzyme is an activation. • Any process that slows or stops the action of an enzyme is an inhibition. • Several strategies usually operate together.
19.7 Enzyme Regulation: Feedback and Allosteric Control • Feedback control occurs when the result of a process feeds information back to affect the beginning of the process. • If D inhibits enzyme 1, the amount of A and B synthesized decreases when no more D is needed. • When more D is needed, enzyme 1 will no longer be inhibited.
19.7 Enzyme Regulation: Feedback and Allosteric Control • In allosteric control, the binding of a molecule (an allosteric regulator or effector) at one site on a protein affects the binding of another molecule at a different site. • Allosteric control can be either positive or negative.
19.8 Enzyme Regulation: Inhibition Reversible Uncompetitive Inhibition • The inhibitor does not compete with the substrate for the active site. It binds to the enzyme-substrate complex so that the reaction occurs less efficiently, or not at all. Reversible Competitive Inhibition • A competitive inhibitor binds reversibly to an active site through noncovalent interactions, but undergoes no reaction, preventing the substrate from entering the active site.
19.8 Enzyme Regulation: Inhibition Irreversible Inhibition • The enzyme’s reaction cannot occur because the substrate cannot connect with the active site. Many irreversible inhibitors are poisons as a result of their ability to completely shut down the active site. • Heavy metal ions, such as mercury and lead, are irreversible inhibitors that form covalent bonds to the sulfur atoms in the —SH groups of cysteine residues.
19.8 Enzyme Regulation: Inhibition Enzyme Inhibitors as Drugs • Angiotensin II, an octapeptide, is a potent pressor—it elevates blood pressure, in part by causing contraction of blood vessels. Angiotensin I, a decapeptide, is an inactive precursor of angiotensin II. To become active, two amino acid residues—His and Leu—must be cut off the end of angiotensin I, a reaction catalyzed by angiotensin-converting enzyme (ACE). • A zinc(II) ion is present in the ACE active site. The extract of venom from a South American pit viper is a mild ACE inhibitor and contains a pentapeptide with a proline residue at the carboxyl-terminal end. • The first ACE inhibitor on the market, captopril, was developed by experimenting with modifications of the proline structure.
19.8 Enzyme Regulation: Inhibition Enzyme Inhibitors as Drugs • Two important AIDS-fighting drugs are enzyme inhibitors. The first, AZT (azidothymidine, also called zidovudine), resembles a molecule essential to reproduction of the AIDS-causing human immunodeficiency virus (HIV). AZT is accepted by an HIV enzyme as a substrate and prevents the virus from producing duplicate copies of itself. • The most successful AIDS drug thus far inhibits a protease, an enzyme that cuts a long protein chain into smaller pieces needed by the HIV. • Protease inhibitors cause dramatic decreases in virus population and symptoms. The success is only achieved by taking a “cocktail” of several drugs including AZT, which requires precise adherence to a schedule of taking 20 pills a day.
19.9 Enzyme Regulation: Covalent Modification and Genetic Control Covalent Modification • Some enzymes are synthesized in inactive form. • Activation of zymogens or proenzymes, requires a chemical reaction that splits off part of the molecule. • Examples of zymogens include trypsinogen, chymostrypsinogen, and proelastase, precursors of enzymes that digest proteins in the small intestine. These enzymes must be inactive when they are synthesized so that they do not immediately digest the pancreas.
19.9 Enzyme Regulation: Covalent Modification and Genetic Control Covalent Modification • Another mode of covalent modification is the reversible addition of phosphoryl groups to serine, tyrosine, or threonine residues. • Kinase enzymes catalyze the addition of a phosphoryl group supplied by ATP (phosphorylation). • Phosphatase enzymes catalyze the removal of the phosphoryl group (dephosphorylation).
19.9 Enzyme Regulation: Covalent Modification and Genetic Control Genetic Control • The synthesis of enzymes, like that of all proteins, is regulated by genes . • The genetic control strategy is especially useful for enzymes needed only at certain stages of development. Mechanisms controlled by hormones can accelerate or decelerate enzyme synthesis.
19.9 Enzyme Regulation: Covalent Modification and Genetic Control Mechanisms of Enzyme Control • Feedback control is exerted on an earlier reactant by a later product in a reaction pathway and is made possible by allosteric control. The feedback molecule binds to a specific enzyme early in the pathway in a way that alters the shape and, therefore, the efficiency of the enzyme. • Inhibition, which is either reversible or irreversible. Reversible inhibition can involve both the substrate and the active site (uncompetitive inhibition) or only the active site (competitive inhibition) by molecules that often mimic substrate structure. Irreversible inhibition occurs because of covalent bonding of the inhibitor to the enzyme. Competitive inhibition is a strategy often utilized in medications, and irreversible inhibition is a mode of action of many poisons. • Production of inactive enzymes (zymogens), which must be activated by cleaving a portion of the molecule. • Covalent modification of an enzyme by addition and removal of a phosphoryl group, with the phosphoryl group supplied by ATP. • Genetic control, whereby the amount of enzyme available is regulated by limiting its synthesis.
19.10 Vitamins and Minerals • Vitamin: An organic molecule, essential in trace amounts that must be obtained in the diet because it is not synthesized in the body. • Water-Soluble Vitamins • Water-soluble vitamins are found in the aqueous environment inside cells. • Water-soluble vitamins contain —OH, —COOH or other polar groups that impart water solubility. • They range from simple molecules like vitamin C to quite large and complex structures like vitamin B12. • Most vitamins are components of larger coenzymes, but vitamin C and biotin are biologically active without any change in structure.
19.10 Vitamins and Minerals • Fat-Soluble Vitamins • The fat-soluble vitamins A, D, E, and K are stored in the body’s fat deposits. • The clinical effects of deficiencies of these vitamins are well documented, but the molecular mechanisms by which they act are not nearly as well understood as those of the water-soluble vitamins. • None has been identified as a coenzyme. • The hazards of overdosing on fat-soluble vitamins are greater than the hazards of overdosing on water-soluble vitamins because the fat-soluble vitamins accumulate in body fats. • Excesses of the water-soluble vitamins are more likely to be excreted in the urine.
19.10 Vitamins and Minerals • Vitamin A is essential for night vision, healthy eyes, and normal development of epithelial tissue. It has three active forms: retinol, retinal, and retinoic acid. It is produced in the body by cleavage of b-carotene.
19.10 Vitamins and Minerals • Vitamin D is related in structure to cholesterol. • It is synthesized when ultraviolet light from the sun strikes a cholesterol derivative in the skin. • In the kidney, vitamin D is converted to a hormone that regulates calcium absorption and bone formation. • Deficiencies are most likely in malnourished individuals living where there is little sunlight.
19.10 Vitamins and Minerals • Vitamin E comprises a group of structurally similar compounds called tocopherols. • Like vitamin C, it is an antioxidant: It prevents the breakdown of vitamin A and polyunsaturated fats by oxidation. • Vitamin E apparently is not toxic in overdosage as are the other fat-soluble vitamins, it is best to avoid excessively large doses of vitamin E.
19.10 Vitamins and Minerals • Vitamin K is a family of structurally related compounds distinguished from each other by hydrocarbon side chains of varying length. • This vitamin is essential to the synthesis of several blood-clotting factors. It is produced by intestinal bacteria, so deficiencies are rare.
19.10 Vitamins and Minerals Vitamins, Minerals, and Food Labels • Much is yet to be learned about the functions of vitamins and minerals in the body, and new information is continuously being reported. • The Food and Nutrition Board of the National Academy of Sciences-National Research Council periodically surveys the latest nutritional information and publishes Recommended Dietary Allowances (RDAs). • The U.S. Food and Drug Administration (FDA), which has among its many responsibilities setting the rules for food labeling. • Since 1994, most packaged food products carry standardized Nutrition Facts labels. • In choosing which vitamins and minerals must be listed on the new labels, the government has focused on those currently of greatest importance in maintaining good health.
19.10 Vitamins and Minerals Antioxidants • An antioxidant is a substance that prevents oxidation. • The dietary antioxidants are vitamin C, vitamin E, and the mineral selenium. • They defuse free radicals, reactive molecular fragments with unpaired electrons which gain stability by picking up electrons from nearby molecules.. • Vitamin E acts by giving up the hydrogen to oxygen-containing free radicals. The hydrogen is then restored by reaction with vitamin C. • Selenium is a cofactor in an enzyme that converts hydrogen peroxide to water before the peroxide can go on to produce free radicals.
19.10 Vitamins and Minerals Minerals • This group of micronutrients is composed primarily, but not entirely, of transition group elements. • A balanced diet supplies sufficient amounts of each of these micronutrients. • Many of the transition elements are necessary for proper functioning of enzymes, since these elements are used as cofactors. • Other minerals are used as building blocks for the body and some exist as electrolytes.
19.10 Vitamins and Minerals • Macrominerals, have required daily amounts greater than 100 mg per day. These include calcium, phosphorous, magnesium, potassium, sodium, chloride, and sulfur. • Adequate, regular intake of calcium and phosphorous is necessary for formation and maintenance of bone. • Magnesium is also necessary for bone metabolism and is stored in bone tissue; it is also a cofactor in many different enzymes. • Sodium, chloride and potassium function as electrolytes, maintaining osmotic balance in both intra- and extracellular spaces.
