© 2013 pearson education, inc. outline 19.1 catalysis by enzymes 19.2 enzyme cofactors 19.3 enzyme...

© 2013 Pearson Education, Inc.

Outline

19.1 Catalysis by Enzymes19.2 Enzyme Cofactors19.3 Enzyme Classification19.4 How Enzymes Work19.5 Effect of Concentration on Enzyme Activity19.6 Effect of Temperature and pH on Enzyme

Activity19.7 Enzyme Regulation: Feedback and Allosteric

Control19.8 Enzyme Regulation: Inhibition19.9 Enzyme Regulation: Covalent Modification

and Genetic Control19.10 Vitamins and Minerals


Goals1. 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.

4. 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.

5. 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.



• 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.



• 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.



• 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.



• 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.



• 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.



• 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.



• 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.



• 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.



• 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.



• 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.



• 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.



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.



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.



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.



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.



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.



• 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.



• 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: InhibitionEnzyme 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: InhibitionEnzyme 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.



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).



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.



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.



• 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.



• 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 -carotene.



• 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.



• 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.



• 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.



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.



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.



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.



• 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.



• The transition elements, chromium, copper, magnesium, manganese, molybdenum, selenium and zinc are classed as micronutrients.– Our bodies need only minute amounts as cofactors for

enzymes.

– Some are highly toxic if ingested in high amounts.

– Because these are transition element cations with variable oxidation states, they can serve as transient holders of electrons during enzymatic reactions.

– Iron is a necessary component of the heme ring present in both myoglobin and hemoglobin, as well as in the cytochromes found in the electron transport system.

– Iodine is essential for synthesis of thyroid hormones, which regulate many functions in the body.


Chapter Summary

1. What are enzymes?

• Enzymes are the catalysts for biochemical reactions.

• They are mostly water-soluble, globular proteins, and many incorporate cofactors, which are either metal ions or the nonprotein organic molecules known as coenzymes.

• One or more substrate molecules (the reactants) enter an active site lined by those protein side chains and cofactors necessary for catalyzing the reaction.

• Six major classes and many subclasses of reactions are catalyzed by enzymes.


Chapter Summary, Continued2. How do enzymes work, and why are they so specific?

• A substrate is drawn into the active site by noncovalent interactions.

• As the substrate enters the active site, the enzyme shape adjusts to best accommodate the substrate and catalyze the reaction (the induced fit).

• Within the enzyme–substrate complex, the substrate is held in the best orientation for reaction and in a strained condition that allows the activation energy to be lowered.

• When the reaction is complete, the product is released and the enzyme returns to its original condition.

• The specificity of each enzyme is determined by the presence within the active site of catalytically active groups, hydrophobic pockets, and ionic or polar groups that exactly fit the chemical makeup of the substrate.


Chapter Summary, Continued

3. What effects do temperature, pH, enzyme concentration, and substrate concentration have on enzyme activity?

• With increasing temperature, reaction rate increases to a maximum and then decreases as the enzyme protein denatures.

• Reaction rate is maximal at a pH that reflects the pH of the enzyme’s site of action in the body.

• In the presence of excess substrate, reaction rate is directly proportional to enzyme concentration.

• With fixed enzyme concentration, reaction rate first increases with increasing substrate concentration and then approaches a fixed maximum at which all active sites are occupied.


Chapter Summary, Continued4. How is enzyme activity regulated?

• The effectiveness of enzymes is controlled by a variety of activation and inhibition strategies.

• A product of a later reaction can exercise feedback control over an enzyme for an earlier reaction in a pathway. Feedback control acts through allosteric control of enzymes that have regulatory sites separate from their active sites. Binding a regulator induces a change of shape in the active site, increasing or decreasing the efficiency of the enzyme.

• Uncompetitive inhibitors act on the enzyme-substrate complex, blocking a second substrate from entering the active site; they lower the maximum reaction rate.

• Competitive inhibitors typically resemble the substrate and reversibly block the active site; they slow the reaction rate but do not change the maximum rate.

• Irreversible inhibitors form covalent bonds to an enzyme that permanently inactivate it; most are poisons.

• Enzyme activity is also regulated by reversible phosphorylation and dephosphorylation, and by synthesis of inactive zymogens that are later activated by removal of part of the molecule.

• Genetic control is exercised by regulation of the synthesis of enzymes.


Chapter Summary, Continued

5. What are vitamins and minerals? • Vitamins are organic molecules required in small amounts in the

diet because our bodies cannot synthesize them.• The water-soluble vitamins are coenzymes or parts of

coenzymes. • The fat-soluble vitamins have diverse and less well understood

functions. • In general, excesses of water-soluble vitamins are excreted and

excesses of fat-soluble vitamins are stored in body fat, making excesses of the fat-soluble vitamins potentially more harmful.

• Vitamin C, β-carotene (a precursor of vitamin A), vitamin E, and selenium work together as antioxidants to protect biomolecules from damage by free radicals.

• Minerals are chemical elements needed in small amounts in the diet. Minerals function as macronutrients (calcium and phosphorous for bone), electrolytes, and micronutrients used primarily as enzyme cofactors.

© 2013 pearson education, inc. outline 19.1 catalysis by enzymes 19.2 enzyme cofactors 19.3 enzyme...

Documents