Chapter 5 Enzymes PDF

Enzymes 5 I. OVERVIEW Virtually all reactions in the body are mediated by enzymes, which are protein catalysts, usually within cells, that increase the rate of reactions without being changed in the overall process. Among the many...

Enzymes 5 I. OVERVIEW Virtually all reactions in the body are mediated by enzymes, which are protein catalysts, usually within cells, that increase the rate of reactions without being changed in the overall process. Among the many biologic reactions that are energetically possible, enzymes selectively channel reactants or substrates, into useful pathways. Thus, enzymes direct all metabolic events. This chapter examines the nature of these catalytic molecules and their mechanisms of action. 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, such as glucosidase and urease. Names of other enzymes include a description of the action performed, for example, lactate dehydrogenase (LDH) and adenylyl cyclase. Some enzymes retain their original trivial names, which give no hint of the associated enzymatic reaction, for example, trypsin and pepsin. B. Systematic name In the systematic naming system, enzymes are divided into six major classes (Fig. 5.1), each with numerous subgroups. For a given enzyme, the suffix - ase is attached to a fairly complete description of the chemical reaction catalyzed, including the names of all the substrates, for example, lactate:nicotinamide adenine dinucleotide (NAD+) oxidoreductase. (Note: Each enzyme is also assigned a classification number. Lactate:NAD+ oxidoreductase is 1.1.1.27.) The systematic names are unambiguous and informative but are frequently too cumbersome to be of general use. 136 Figure 5.1 The six major classes of enzymes with examples. NAD(H), nicotinamide adenine dinucleotide; THF, tetrahydrofolate; CoA, coenzyme A; CO2, carbon dioxide; NH3, ammonia; ADP, adenosine diphosphate; Pi, inorganic phosphate. 137  Figure 5.2 Schematic representation of an enzyme with one active site binding a substrate molecule. Potentially confusing enzyme nomenclature includes enzymes with similar names but different functions or mechanisms. For example, synthetases require ATP, while synthases do not require ATP. Phosphatases use water to remove a phosphate group, while phosphorylases use inorganic phosphate to break a bond and generate a phosphorylated product. Dehydrogenases (using NAD+ or flavin adenine dinucleotide, FAD) accept electrons in a redox reaction. Oxidases use oxygen as the acceptor, with no oxygen atoms incorporated into the substrate, while oxygenases do incorporate oxygen atoms into their substrates. III. PROPERTIES An enzyme is an efficient, specific protein catalyst that combines with a substrate at the enzyme active site and performs chemistry on that substrate to convert it to product. Without enzymes most biochemical reactions would not occur quickly enough to have physiologic importance in the human body. While enzymes increase the velocity of a chemical reaction they are not consumed during the reaction. (Note: Some ribonucleic acids [RNAs] can catalyze reactions that affect phosphodiesterase and peptide bonds. RNAs with catalytic activity are called ribozymes and are much less common than protein catalysts.) A. Active site 138  Enzyme molecules contain a special pocket or cleft called the active site which is formed by folding of the protein. The active site contains amino acid residues whose side chains participate in substrate binding and catalysis (Fig. 5.2). The substrate first binds the enzyme, forming an enzyme– substrate (ES) complex. Binding is thought to cause a conformational change in the enzyme (induced fit model) that allows a rapid conversion of the ES to enzyme–product (EP) complex that subsequently dissociates to free enzyme and product. B. Efficiency Enzyme-catalyzed reactions are highly efficient, proceeding from 103 to 108 times faster than uncatalyzed reactions. The number of substrate molecules converted to product per enzyme molecule per second is called the turnover number, or kcat, and typically is 102 to 104 second−1. (Note: kcat is the rate constant for the conversion of ES to E + P.) C. Specificity Enzymes are highly specific and are capable of interacting with one or a very few substrates and can catalyze only one type of chemical reaction. The set of enzymes synthesized within a cell determines which reactions occur in that cell. 139  Figure 5.3 The intracellular location of some important biochemical pathways. TCA, tricarboxylic acid; PP, pentose phosphate. D. Holoenzymes, apoenzymes, cofactors, and coenzymes Some enzymes require nonprotein components to have enzymatic activity. The term holoenzyme refers to the protein component of the enzyme along with its nonprotein component, whereas the enzyme without its nonprotein moiety is termed an apoenzyme and is inactive. For enzymes that require nonprotein components, those components must be present for the enzyme to function in catalysis. 140  If the nonprotein moiety is a metal ion, such as zinc (Zn2+) or iron (Fe2+), it is called a cofactor. If it is a small organic molecule, it is termed a coenzyme. Coenzymes or cosubstrates only transiently associate with the enzyme and dissociate from the enzyme in an altered state (for example, NAD+). If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group (for example, FAD). Coenzymes commonly are derived from vitamins. For example, NAD+ contains niacin, and FAD contains riboflavin. E. Regulation Enzyme activity can often be increased or decreased, so that the rate of product formation responds to the present cellular needs. F. Location within the cell Most enzymes function inside cells, within the confines of plasma membranes. Many enzymes are localized in specific organelles within the cell (Fig. 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. MECHANISM OF ENZYME ACTION 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 activation energy (Ea), is the energy difference between that of the reactants and a high-energy intermediate, the transition state (T*), which is formed during the conversion of reactant to product. 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. A ⇄ T* ⇄ B 141 1. Activation energy: The peak of energy in Figure 5.4 is the difference in free energy between the reactant and T*, in which the high-energy, short- lived intermediate is formed during the conversion of reactant to product. Because of the high Ea, the rates of uncatalyzed chemical reactions are often slow. Figure 5.4 Effect of an enzyme on the activation energy (Ea) of a reaction. ΔG, change in free energy. 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 142  between reactant and product. The rate of reaction is determined by the number of such energized molecules. In general, the lower the Ea, the more molecules have sufficient energy to pass through the transition state and, therefore, 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 Ea (see Fig. 5.4). The enzyme does not change the free energies of the reactants (substrates) or products and, therefore, does not change the equilibrium of the reaction. It does, however, accelerate the rate by which equilibrium is reached. B. Active site chemistry The active site is not a passive receptacle for binding the substrate but, rather, is a complex molecular machine that can employs diverse 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 examples. 1. Transition-state stabilization: The active site often acts as a flexible molecular template that binds the substrate and initiates its conversion to the transition state, a structure in which the bonds are not like those in the substrate or the product (see T* at the top of the curve in Fig. 5.4). By stabilizing the transition state, the enzyme greatly increases the concentration of the reactive intermediate that can be converted to product and, thus, accelerates the reaction. (Note: The transition state cannot be isolated.) 143 144  Figure 5.5 Schematic representation of energy changes accompanying formation of an enzyme–substrate complex and subsequent formation of a transition state. 2. Catalysis: 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 ES 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 loses (general acid) protons, mediated by the pK of histidine in proteins being close to physiologic pH. Serine at the active site forms a transient covalent bond with the substrate. 3. Transition-state visualization: The enzyme-catalyzed conversion of substrate to product can be depicted as being similar to removing a sweater (chemical group) from an uncooperative infant (substrate) (Fig. 5.5). The process has a high Ea because the only reasonable strategy for removing the garment requires that both arms being fully extended over the head, an unlikely posture to be adopted without a catalyst. We can envision a parent acting as an enzyme, first coming in contact with the baby (forming ES) and then guiding the baby’s arms into an extended, vertical position, analogous to the transition state. This posture (conformation) of the baby facilitates the removal of the sweater, forming the disrobed baby, which represents 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 145 Enzymes can be isolated from cells and their properties studied in a test tube, that is, in vitro. Different enzymes show different responses to changes in substrate concentration, temperature, and pH. This section describes factors that influence the reaction velocity of enzymes. Enzymatic responses to these factors give us valuable clues as to how enzymes function in living cells, that is, in vivo. 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 μmol of product formed per second. The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached (Fig. 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. Figure 5.6 Effect of substrate concentration on reaction velocity. 2. Shape of the enzyme kinetics curve: Most enzymes follow Michaelis– Menten kinetics (see p. 62), in which a plot of initial reaction velocity (vo) against substrate concentration is hyperbolic (similar in shape to that of the oxygen-dissociation curve of myoglobin; see Chapter 3). In contrast, allosteric enzymes do not follow Michaelis–Menten kinetics and instead show a sigmoidal curve (see Fig. 5.6) that is similar in shape to the oxygen-dissociation curve of hemoglobin. 146 B. Temperature 1. Velocity increase with temperature: The reaction velocity increases with temperature until a peak velocity is reached (Fig. 5.7). This increase is the result of the increased number of substrate molecules having sufficient energy to pass over the energy barrier and form the products of the reaction. 2. Velocity decrease with higher temperature: Further elevation of the temperature causes a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme (see Fig. 5.7). Figure 5.7 Effect of temperature on an enzyme-catalyzed reaction. Normal body temperature is 37°C. The optimum temperature for most human enzymes is between 35° and 40°C. Human enzymes start to denature at temperatures above 40°C, but thermophilic bacteria found in hot springs have optimum temperatures of 70°C. C. pH 1. pH effect on active site ionization: The concentration of protons ([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 147  enzyme be in the protonated form (−NH3+). Because this group is deprotonated at alkaline pH, the rate of the reaction declines. 2. pH effect 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. Variable pH optimum: 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 (Fig. 5.8). Figure 5.8 Effect of pH on enzyme-catalyzed reactions. VI. MICHAELIS–MENTEN KINETICS In a paper published in 1913, Leonor Michaelis and Maud Menten proposed a model that accounts for most features of many enzyme-catalyzed reactions. In this model, the enzyme reversibly combines with its substrate to form an ES complex that subsequently yields product, regenerating the free enzyme. The reaction model, involving one substrate molecule, is represented below: 148  Where S is the substrate; E is the enzyme; ES is the enzyme–substrate complex; P is the product; k1, k−1, and k2 (or, kcat) are rate constants. A. Michaelis–Menten equation The Michaelis–Menten equation describes how reaction velocity varies with substrate concentration: Where vo = initial reaction velocity; Vmax = maximal velocity = kcat [E]Total; Km = Michaelis constant = (k−1 + k2)/k1; [S] = substrate concentration; The following assumptions are made in deriving the Michaelis–Menten rate equation. 1. Enzyme and substrate relative concentrations: The substrate concentration ([S]) is much greater than the concentration of enzyme so that the percentage of total substrate bound by the enzyme at any one time is small. 2. Steady-state assumption: The concentration of the ES complex does not change with time (the steady-state assumption), that is, the rate of formation of ES is equal to that of the breakdown of ES (to E + S and to E + P). In general, an intermediate in a series of reactions is said to be in steady state when its rate of synthesis is equal to its rate of degradation. 3. Initial velocity: Initial reaction velocities (vo) are used in the analysis of enzyme reactions. This means that the rate of the reaction is measured as soon as enzyme and substrate are mixed. At that time, the concentration of product is very small, and therefore, the rate of the reverse reaction from product to substrate can be ignored. B. Important conclusions 149 1. Km characteristics: Km, the Michaelis constant, is characteristic of an enzyme and its particular substrate and reflects the affinity of the enzyme for that substrate. Km is numerically equal to the substrate concentration at which the reaction velocity is equal to one half Vmax. Km does not vary with enzyme concentration. a. Small Km: A numerically small (low) Km reflects a high affinity of the enzyme for substrate, because a low concentration of substrate is needed to half-saturate the enzyme—that is, to reach a velocity that is one half Vmax (Fig. 5.9). b. Large Km: A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration of substrate is needed to half saturate the enzyme. 2. Velocity relationship to enzyme concentration: The rate of the reaction is directly proportional to the enzyme concentration because [S] is not limiting. For example, if the enzyme concentration is halved, the initial rates of the reaction (vo) and that of Vmax are reduced to half that of the original. 150  Figure 5.9 Effect of substrate concentration on reaction velocities for two enzymes: enzyme 1 with a small Michaelis constant (Km) and enzyme 2 with a large Km. Vmax, maximal velocity. 3. Reaction order: When [S] is much less () than Km, the velocity is constant and equal to Vmax. The rate of reaction is then independent of substrate concentration because the enzyme is saturated with substrate and is said to be zero order with respect to substrate concentration (see Fig. 5.10). C. Lineweaver–Burk plot When vo is plotted against [S], it is not always possible to determine when Vmax has been achieved because of the gradual upward slope of the hyperbolic curve at high substrate concentrations. However, as Hans 151 Lineweaver and Dean Burk first described in 1934, if 1/vo is plotted versus 1/[S], then a straight line is obtained (Fig. 5.11). This plot, the Lineweaver– Burk plot, also called a double-reciprocal plot, can be used to calculate Km and Vmax as well as to determine the mechanism of action of enzyme inhibitors. The equation describing the Lineweaver–Burk plot is: where the intercept on the x axis is equal to − 1/Km, and the intercept on the y axis is equal to 1/Vmax. (Note: The slope = Km/Vmax.) 152  Figure 5.10 Effect of substrate concentration on reaction velocity for an enzyme-catalyzed reaction. Vmax, maximal velocity; Km, Michaelis constant. VII. ENZYME INHIBITION Any substance that can decrease the velocity of an enzyme-catalyzed reaction is considered to be an inhibitor. Inhibitors can be reversible or irreversible. Irreversible inhibitors bind to enzymes through covalent bonds. Lead, for example, can act as an irreversible inhibitor of some enzymes. It forms covalent bonds with the sulfhydryl side chain of cysteine in proteins. Ferrochelatase, an enzyme involved in heme synthesis, is irreversibly inhibited by lead. Reversible inhibitors bind to enzymes through noncovalent bonds forming an enzyme– 153 inhibitor complex. Dilution of the enzyme–inhibitor complex results in dissociation of the reversibly bound inhibitor and recovery of enzyme activity. The two most commonly encountered types of reversible inhibition are competitive and noncompetitive. A. Competitive inhibition This type of inhibition occurs when the inhibitor binds reversibly to the same site that the substrate would normally occupy and, therefore, competes with the substrate for binding to the enzyme active site. 1. Effect on Vmax: The effect of a competitive inhibitor is reversed by increasing the concentration of substrate. At a sufficiently high [S], the reaction velocity reaches the Vmax observed in the absence of inhibitor, that is, Vmax is unchanged in the presence of a competitive inhibitor (Fig. 5.12). 2. Effect on Km: A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve half Vmax. Figure 5.11 Lineweaver–Burk plot. vo, initial reaction velocity; Vmax, maximal velocity; Km, Michaelis constant; [S], substrate concentration. 3. Effect on the Lineweaver–Burk plot: Competitive inhibition shows a characteristic Lineweaver–Burk plot in which the plots of the inhibited and uninhibited reactions intersect on the y axis at 1/Vmax (Vmax is unchanged). The inhibited and uninhibited reactions show different x-axis intercepts, indicating that the apparent Km is increased in the presence of the competitive inhibitor because − 1/Km moves closer to zero from a 154  negative value (see Fig. 5.12). (Note: An important group of competitive inhibitors are the transition state analogs, stable molecules that approximate the structure of the transition state, and, therefore, bind the enzyme more tightly than does the substrate.) Figure 5.12 A: Effect of a competitive inhibitor on the reaction velocity versus substrate concentration ([S]) plot. B: Lineweaver–Burk plot of competitive inhibition of an enzyme. (Note: The slope increases if inhibitor concentration increases.) 4. Statin drugs as examples of competitive inhibitors: Statin drugs are cholesterol-lowering agents that competitively inhibit the rate-limiting (slowest) step in cholesterol biosynthesis. This reaction is catalyzed by hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase; see Chapter 19). Statins, such as atorvastatin and pravastatin, are structural analogs of the natural substrate for this enzyme and compete effectively to inhibit HMG CoA reductase. By doing so, they inhibit de novo cholesterol synthesis (Fig. 5.13). 155  Figure 5.13 Pravastatin competes with hydroxymethylglutaryl coenzyme A (HMG CoA) for the active site of HMG CoA reductase. B. Noncompetitive inhibition This type of inhibition is recognized by its characteristic effect causing a decrease in Vmax (Fig. 5.14). Noncompetitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme. The noncompetitive inhibitor can bind either free enzyme or the ES complex, thereby preventing the reaction from occurring (Fig. 5.15). 1. Effect on Vmax: Effects of a noncompetitive inhibitor cannot be overcome by increasing the concentration of substrate. Therefore, noncompetitive inhibitors decrease the apparent Vmax of the reaction. 156  Figure 5.14 A: Effect of a noncompetitive inhibitor on the reaction velocity versus substrate concentration ([S]) plot. B: Lineweaver–Burk plot of noncompetitive inhibition of an enzyme. (Note: The slope increases if inhibitor concentration increases.) 2. Effect on Km: Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme. Therefore, the enzyme shows the same Km in the presence or absence of the noncompetitive inhibitor, that is, Km is unchanged in the presence of a noncompetitive inhibitor. 3. Effect on Lineweaver–Burk plot: Noncompetitive inhibition is readily differentiated from competitive inhibition by plotting 1/vo versus 1/[S] and noting that the apparent Vmax decreases in the presence of a noncompetitive inhibitor, whereas Km is unchanged (see Fig. 5.14). C. Enzyme inhibitors as drugs At least half of the 10 most commonly prescribed drugs in the United States act as enzyme inhibitors. For example, the widely prescribed β-lactam antibiotics, such as penicillin and amoxicillin, act by inhibiting enzymes involved in bacterial cell wall synthesis. Drugs may also act by inhibiting extracellular reactions. This is illustrated by angiotensin-converting enzyme (ACE) inhibitors. They lower blood pressure by blocking plasma ACE that cleaves angiotensin I to form the potent vasoconstrictor, angiotensin II. These drugs, which include captopril, enalapril, and lisinopril, cause vasodilation and, therefore, a reduction in blood pressure. Aspirin, a nonprescription drug, irreversibly inhibits prostaglandin and thromboxane synthesis by inhibiting cyclooxygenase. 157  Figure 5.15 A noncompetitive inhibitor binding to both free enzyme and enzyme– substrate (ES) complex. VIII. ENZYME REGULATION The regulation of the reaction velocity of enzymes is essential if an organism is to coordinate its numerous metabolic processes. The rates of most enzymes are responsive to changes in substrate concentration, because the intracellular level of many substrates is in the range of the Km. Thus, an increase in substrate concentration prompts an increase in reaction rate, which tends to return the concentration of substrate toward normal. In addition, some enzymes with specialized regulatory functions respond to allosteric effectors and/or covalent modification or they show altered rates of enzyme synthesis (or degradation) when physiologic conditions are changed. A. Allosteric enzymes Allosteric enzymes do not follow Michaelis–Menten kinetics but are regulated by molecules called effectors that bind to them noncovalently at a site other than the active site. These enzymes are almost always composed of multiple subunits, and the regulatory (allosteric) site that binds the effector is distinct from the substrate-binding site and may be located on a subunit that is not itself catalytic. 158 Effectors that inhibit enzyme activity are termed negative effectors, whereas those that increase enzyme activity are called positive effectors. Positive and negative effectors can affect the affinity of the enzyme for its substrate (K0.5), modify the maximal catalytic activity of the enzyme (Vmax), or both (Fig. 5.16). Note that allosteric enzymes frequently catalyze the committed step, often the rate-limiting step, early in a pathway. Figure 5.16 Effects of negative or positive effectors on an allosteric enzyme. A: Maximal velocity (Vmax) is altered. B: The substrate concentration that gives half maximal velocity (K0.5) is altered. 159  1. Homotropic effectors: When the substrate itself serves as an effector, the effect is said to be homotropic, or same as the substrate. Most often, an allosteric substrate functions as a positive effector. In such a case, the presence of a substrate molecule at one site on the enzyme enhances the catalytic properties of the other substrate-binding sites. That is, their binding sites cooperate with each other for substrate binding and are said to exhibit cooperativity. These enzymes show a sigmoidal curve when vo is plotted against substrate concentration, as shown in Figure 5.16. This contrasts with the hyperbolic curve characteristic of enzymes following Michaelis–Menten kinetics, as previously discussed. (Note: The concept of cooperativity of substrate binding is analogous to the binding of oxygen to hemoglobin [see Chapter 3].) 2. Heterotropic effectors: When the effector is a different molecule than the substrate, it is said to be heterotropic. For example, consider the feedback inhibition shown in Figure 5.17. The enzyme that converts D to E has an allosteric site that binds the end product, G. If the concentration of G increases (e.g., because it is not used as rapidly as it is synthesized), the first irreversible step unique to the pathway is typically inhibited. Feedback inhibition provides the cell with appropriate amounts of a product it needs by regulating the flow of substrate molecules through the pathway that synthesizes that product. Heterotropic effectors are commonly encountered. For example, the glycolytic enzyme phosphofructokinase-1 is allosterically inhibited by citrate, which is not a substrate for the enzyme. Figure 5.17 Feedback inhibition of a metabolic pathway. B. Covalent modification Many enzymes are regulated by covalent modification, most often by the addition or removal of phosphate groups from specific serine, threonine, or 160  tyrosine residues of the enzyme. Protein phosphorylation is recognized as one of the primary ways in which cellular processes are regulated. 1. Phosphorylation and dephosphorylation: Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that catalyze the addition of a phosphate group to its protein or enzyme substrate, using ATP as the phosphate donor. Phosphoprotein phosphatases are enzymes that cleave phosphate groups from phosphorylated proteins and enzymes (Fig. 5.18). Figure 5.18 Covalent modification by the addition and removal of phosphate groups. (Note: HPO42− may be represented as Pi and PO32− as P.) ADP, adenosine diphosphate. 2. Enzyme response to phosphorylation: Depending on the specific enzyme, the phosphorylated form of an enzyme may be more or less active than the unphosphorylated enzyme. For example, hormone- mediated phosphorylation of glycogen phosphorylase, an enzyme that degrades glycogen, increases its activity, whereas phosphorylation of glycogen synthase, an enzyme that synthesizes glycogen, decreases its activity (see Chapter 11). C. Enzyme synthesis The regulatory mechanisms described above can modify the activity of existing enzyme molecules. However, cells can also regulate the amount of enzyme present by altering the rate of enzyme degradation or, more typically, the rate of enzyme synthesis. The increase (induction) or decrease (repression) of enzyme synthesis leads to an alteration in the total population of active sites. Enzymes subject to regulation of synthesis are often those 161  that are needed at only one stage of development or under selected physiologic conditions. For example, elevated levels of insulin as a result of high blood glucose levels cause an increase in the synthesis of key enzymes involved in glucose metabolism (see Chapter 23). In contrast, enzymes that are in constant use are usually not regulated by altering the rate of enzyme synthesis. Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days), compared with allosterically or covalently regulated changes in enzyme activity, which occur in seconds to minutes. Table 5.1 summarizes the common ways that enzyme activity is regulated. Table 5.1 Mechanisms for Regulating Enzyme Activity Regulator Event Typical Effector Results Time Required for Change Substrate availability Substrate Change in velocity Immediate (Vo) Product inhibition Reaction product Change in Vmax Immediate and/or Km Allosteric control Pathway end product Change in Vmax Immediate and/or K0.5 Covalent modification Another enzyme Change in Vmax Immediate to minutes and/or Km Synthesis or Hormone or Change in the Hours to days degradation of metabolite amount of enzyme enzyme Note: Inhibition by pathway end product is also referred to as feedback inhibition. IX. ENZYMES IN HUMAN BLOOD While most enzymes function intracellularly, enzymes can be found outside cells in fluids including blood plasma, the fluid portion of blood. Enzymes that appear in blood plasma of healthy persons can be classified into two major groups. First, a relatively small group of enzymes are actively secreted into the blood by certain cell types. For example, the liver secretes zymogens (inactive precursors) of the protease enzymes involved in blood coagulation. Such proteases can be activated and have enzymatic function in the blood. Second, enzymes are released from cells during normal cell turnover. These enzymes almost always function only intracellularly and have no ability to catalyze reactions in blood plasma. In healthy individuals, the levels of these enzymes are fairly constant and represent a steady state in which the rate of release from damaged cells into the plasma is balanced by an equal rate of removal from the 162 plasma. Increased blood plasma levels of these enzymes may indicate tissue damage and cell death that is greater than cell death from normal turnover (Fig. 5.19). Blood plasma is the fluid, noncellular fraction of blood. Laboratory assays of enzyme activity most often use serum, which is the fluid obtained by centrifugation of whole blood after it has been allowed to coagulate. Plasma is a physiologic fluid, whereas serum is a fluid prepared in the laboratory from a patient’s whole blood sample. A. Blood plasma enzyme levels in disease states Many diseases cause tissue damage that includes the rupture of plasma membranes and lysis of cells in the tissue. As a result, the damaged cells release their contents into fluids, including the blood plasma, causing an increased concentration of the enzymes in the plasma. These enzymes are normally intracellular and cannot catalyze reactions when present outside their normal cellular location. However, these enzymes are routinely measured in patient’s blood samples for diagnostic purposes. The level of specific enzyme activity in the plasma frequently correlates with the extent of tissue damage. Therefore, determining the extent of elevation of a particular enzyme activity in the blood plasma is often useful in evaluating the extent of tissue damage, response to therapies, and the prognosis for the patient. Figure 5.19 Release of enzymes from normal (A) and diseased or traumatized (B) cells. 163  Table 5.2 Some Clinically Useful Enzymes Enzyme Abbreviation Main Tissue Useful to Assess Source(s) Alanine ALT Liver Liver damage or disease aminotransferase Alkaline ALP Liver, bone Liver and bone diseases phosphatase Amylase Amylase Pancreas Pancreatic diseases Aspartate AST Liver, muscle Liver and muscle aminotransferase diseases Creatine kinase CK Muscle Muscle damage or disease Gamma glutamyl GGT Liver, bile duct Hepatobiliary disease transferase (obstructive jaundice) Lipase Lipase Pancreas Pancreatic diseases Lactate LDH Red blood cells, General marker of cell dehydrogenase liver, muscle—most death; particularly in cells hemolysis, hepatic or muscle diseases 5’ Nucleotidase 5’NT Liver Hepatobiliary disease (obstructive jaundice) Appearance of these enzymes in blood can indicate damage to cells in the tissue where the enzyme normally functions. B. Plasma enzymes as diagnostic tools Some enzymes show relatively high activity in only one or a few tissues (Table 5.2). Therefore, the presence of increased levels of these enzymes in blood plasma reflects damage to the corresponding tissue. For example, the enzyme alanine aminotransferase (ALT) is one of many enzymes that are abundant in the liver. The appearance of elevated levels of ALT in plasma signals possible damage to hepatic tissue. Measurement of ALT released into a patient’s blood from dying cells is part of the liver function test panel. Increases in plasma levels of enzymes with a wide tissue distribution provide a less specific indication of the site of cellular injury and limits their diagnostic value. C. Isoenzymes Isoenzymes are variant forms of a particular enzyme that all catalyze the same reaction but have slightly different physical properties because of genetically determined differences in amino acid sequence. For this reason, isoenzymes may contain different numbers of charged amino acids, which 164 allows them to be separated from each other by electrophoresis (the movement of charged particles in an electric field) (Fig. 5.20). Different organs commonly contain characteristic proportions of different isoenzymes. LDH is found in relatively high concentration in most tissues; five isoenzyme forms of LDH exist, LD 1–5, with LD5 prevalent in liver and skeletal muscle, LD2 in red blood cells and LD1 in myocardial muscle for example. The pattern of isoenzymes found in the blood plasma may, therefore, serve as a means of identifying the site of tissue damage. The plasma levels of various isoenzyme forms of LDH and of creatine kinase (CK) vary under different disease states. 1. Isoenzyme quaternary structure: Isoenzymes of a given enzyme often contain different subunits in various combinations. For example, LDH occurs as five isoenzymes and each exists as a tetramer, containing four subunits (combinations of subunits called H and M for heart and skeletal muscle where they were first discovered) such that LD1 = HHHH, LD2 (HHHM), LD3 (HHMM), LD4 (HMMM), and LD5 (MMMM). CK occurs as three isoenzymes. Each CK isoenzyme is a dimer composed of two polypeptide subunits (called B and M subunits for brain and skeletal muscle) associated in one of three combinations: CK1 = BB, CK2 = MB, and CK3 = MM. Each CK isoenzyme shows a characteristic electrophoretic mobility (see Fig. 5.20). (Note: Virtually all CK in the brain is the BB isoform, whereas it is MM in skeletal muscle. In cardiac muscle, there a majority of CK is MM, but the presence of CK MB is unique to myocardium.) 2. Historical use in diagnosis of myocardial infarction: Measurement of blood levels of isoenzymes with cardiac specificity (biomarkers) had an important use in the diagnosis of MI prior to the advent of testing for cardiac proteins known as troponins (see below). Because myocardial muscle is the only tissue that contains >5% of the total CK activity as the CK MB (CK2) isoenzyme, its appearance in blood plasma is virtually specific for damage to myocardial muscle and is seen after an acute myocardial infarction (MI or heart attack). Following an acute MI, CK MB appears in a patient’s blood plasma within 4 to 8 hours following onset of chest pain, reaches a peak of activity at ∼24 hours, and returns to baseline after 48 to 72 hours (Fig. 5.21). 165 166  Figure 5.20 Subunit composition, electrophoretic mobility, and enzyme activity of creatine kinase (CK) isoenzymes. Clinical Application 5.1: Diagnostic Use of Troponins Troponins T (TnT) and I (TnI) are regulatory proteins involved in muscle contractility. Cardiac-specific isoforms (cTn) of troponins are released into the plasma in response to cardiac damage. and there is a highly sensitive and specific indication of damage to cardiac tissue. cTn appear in plasma within 4 to 6 hours after an MI, peak in 24 to 36 hours, and remain elevated for 3 to 10 days. Elevated cTn, in combination with the clinical presentation and characteristic changes in the ECG, are currently considered the “gold standard” in the diagnosis of an MI. While the appearance characteristics of cTN in blood plasma after an acute MI are similar to those of CK MB, the change from baseline to peak values is much greater for cTN (see Fig. 5.21). 167 Figure 5.21 Appearance of creatine kinase isozyme CK-MB and cardiac troponin in plasma after an myocardial infarction. (Note: Either cardiac troponin T or I may be measured.) 168 X. Chapter Summary Enzymes are protein catalysts that increase the velocity of a chemical reaction by providing an alternate reaction pathway with a lower activation energy (Fig. 5.22). Enzymes contain a specialized cleft called the active site, which binds the substrate, forming an ES complex, with conversion to product (ES → EP → E + P). Most enzymes show Michaelis–Menten kinetics, and a plot of the initial reaction velocity (vo) against [S] has a hyperbolic shape; allosteric enzymes show a sigmoidal curve. A Lineweaver–Burk or double-reciprocal plot of 1/v and 1/[S] transforms the hyperbolic shaped curve to a straight line and allows easier determination of Vmax (maximal velocity) and Km (Michaelis constant, which reflects affinity for substrate). An inhibitor is any substance that can decrease the velocity of an enzyme- catalyzed reaction. The two most common types of enzyme inhibition are competitive, which increase the apparent Km and noncompetitive which decreases the apparent Vmax. Allosteric enzymes are composed of subunits and are regulated by effectors that bind noncovalently at a site other than the active site. Positive allosteric effectors increase enzyme activity and negative effectors decrease enzyme activity. Enzymes can also be regulated by covalent modification, most often via phosphorylation catalyzed by protein kinases while phosphoprotein phosphatases remove phosphate groups. Regulation can also occur by changes in the rate of synthesis or degradation. Since most enzymes function intracellularly, their appearance in blood plasma can indicate damage to a corresponding tissue, giving enzymes a diagnostic value in medicine. 169