UNIT I: Protein Structure and Function CHAPTER 5:

UNIT I: Protein Structure and Function CHAPTER 5: ENZYMES PART 2 VI. MICHAELIS-MENTEN KINETICS A. Reaction model Leonor Michaelis and Maude Menten proposed a simple model that accounts for most of the features of enzyme catalyzed reactions. The model, involving one substrate molecule, is represented below: where S is the substrate

E is the enzyme ES is the enzymesubstrate complex P is the product k1, k-1 , and k2 are rate constants 2 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 In this model, the enzyme reversibly combines with its substrate to form an ES complex that subsequently yields product, regenerating the free enzyme. VI. MICHAELIS-MENTEN KINETICS B. Michaelis -Menten equation

where Vo = initial reaction velocity Vmax = maximal velocity Km = Michaelis constant = (k-1 + k2)/k1 [S] = substrate concentration 3 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 The Michaelis Menten equation describes how reaction velocity varies with substrate concentration: VI. MICHAELIS-MENTEN KINETICS B. Michaelis -Menten equation The following assumptions are made in deriving the Michaelis Menten rate equation:

1. Relative concentrations of enzyme and substrate: The concentration of substrate ([S]) is much greater than the concentration of enzyme ([E]), so that the percentage of total substrate bound by the enzyme at any one time is small. [ES] 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 4 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 2. Steady-state assumption: VI. MICHAELIS-MENTEN KINETICS

B. Michaelis -Menten equation 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 back reaction from product to substrate can be ignored 5 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 3. Initial velocity: VI. MICHAELIS-MENTEN KINETICS

C. Important conclusions 1. Characteristics of Km: 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 enzymethat is, to reach a velocity that is 12V max (Figure 5.9). b) Large Km: 6 A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration substrate 2016 is needed to half saturate the enzyme. Dr. M. of Alzaharna

Dr. M. Alzaharna 2016 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 12Vmax. Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 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.

VI. MICHAELIS-MENTEN KINETICS C. Important conclusions 2. Relationship of velocity to enzyme concentration: The rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations. For example, if the enzyme concentration is halved, the initial rate of the reaction (vo), as well as that of Vmax, are reduced to half that of the original. When [S] is much less than Km, the velocity of the reaction is approximately proportional to the substrate concentration (Figure 5.10). The rate of reaction is then said to be first order with respect to substrate. When [S] is much greater than Km, the velocity is constant and equal to Vmax. The rate of reaction is then independent of substrate concentration (the enzyme is saturated with substrate) and is said to be zero order with respect to substrate concentration (see Figure 5.10) 8

Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 3. Order of reaction: Dr. M. Alzaharna 2016 Figure 5.10: Effect of substrate concentration on reaction velocity for an enzyme-catalyzed reaction. Vmax = maximal velocity; Km = Michaelis constant. Dr. M. Alzaharna 2016 VI. MICHAELIS-MENTEN KINETICS D. 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. 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: 10 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 However, if 1/vo is plotted versus 1/[S], a straight line is obtained (Figure 5.11). Dr. M. Alzaharna 2016

Dr. M. Alzaharna 2016 Figure 5.11: Lineweaver-Burk plot. vo = reaction velocity; Vmax = maximal velocity; Km = Michaelis constant; [S] = substrate concentration. VII. INHIBITION OF ENZYME ACTIVITY Any substance that can decrease the velocity of an enzyme catalyzed reaction is called an inhibitor. Inhibitors can be reversible or irreversible. Irreversible inhibitors bind to enzymes through covalent bonds. Ferrochelatase, an enzyme involved in heme synthesis, is irreversibly inhibited by lead. Reversible inhibitors bind to enzymes through noncovalent bonds and, thus, dilution of the enzymeinhibitor 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. 12 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 Lead, for example, forms covalent bonds with the sulfhydryl side chain of cysteine in proteins. VII. INHIBITION OF ENZYME ACTIVITY 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 that site. The effect of a competitive inhibitor is reversed by increasing [S]. At a sufficiently high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of inhibitor (Figure 5.12).

2. Effect on Km: A competitive inhibitor increases the apparent K m for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve 12Vmax. 13 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 1. Effect on Vmax: Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 Figure 5.12: A. Effect of a competitive inhibitor on the reaction velocity

versus substrate ([S]) plot. B. Lineweaver-Burk plot of competitive inhibition of an enzyme. VII. INHIBITION OF ENZYME ACTIVITY A. Competitive inhibition 3. Effect on the Lineweaver-Burk plot: Competitive inhibition shows a characteristic LineweaverBurk plot in which the plots of the inhibited and uninhibited reactions intersect on the y axis at 1/V max (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 negative value (see Figure 5.12). 15 This group of antihyperlipidemic agents competitively inhibits the rate limiting (slowest) step in cholesterol biosynthesis. This reaction is catalyzed by hydroxymethylglutarylCoA reductase (HMG-CoA

reductase). Statins, such as atorvastatin (Lipitor) and pravastatin (Pravachol), 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, thereby lowering plasma cholesterol levels (Figure 5.13). Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 4. Statin drugs as examples of competitive inhibitors: Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 Figure 5.13: Pravastatin competes with HMG-CoA for the active site of HMG-CoA

reductase. HMG-CoA = hydroxymethylglutaryl coenzyme A. VII. INHIBITION OF ENZYME ACTIVITY B. Noncompetitive inhibition This type of inhibition is recognized by its characteristic effect on Vmax (Figure 5.14). The noncompetitive inhibitor can bind either free enzyme or the enzyme-substrate complex, thereby preventing the reaction from occurring (Figure 5.15). 17 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016

Noncompetitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme. Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 Figure 5.14: A. Effect of a noncompetitive inhibitor on the reaction velocity versus substrate ([S]) plot. B. Lineweaver-Burk plot of noncompetitive inhibition of an enzyme. Dr. M. Alzaharna 2016 Figure 5.15: A noncompetitive inhibitor binding to both free enzyme and enzyme substrate (ES) complex.

Dr. M. Alzaharna 2016 VII. INHIBITION OF ENZYME ACTIVITY B. Noncompetitive inhibition 1. Effect on Vmax: 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. 20 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 Noncompetitive inhibition cannot be overcome by increasing

the concentration of substrate. Therefore, noncompetitive inhibitors decrease the apparent Vmax of the reaction. VII. INHIBITION OF ENZYME ACTIVITY B. Noncompetitive inhibition 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 Figure 5.14). Note: Oxypurinol, a metabolite of the drug allopurinol, is a noncompetitive inhibitor of xanthine oxidase, an enzyme of purine degradation. 21 Dr. M. Alzaharna 2016

Dr. M. Alzaharna 2016 3. Effect on Lineweaver-Burk plot: VII. INHIBITION OF ENZYME ACTIVITY C. Enzyme inhibitors as drugs At least half of the ten most commonly prescribed drugs in the United States act as enzyme inhibitors. For example, the widely prescribed -lactam antibiotics, such as penicillin and amoxicillin, act by inhibiting enzymes involved in bacterial cell wall synthesis. This is illustrated by angiotensin-converting enzyme (ACE) inhibitors. They lower blood pressure by blocking the enzyme that cleaves angiotensin I to form the potent vasoconstrictor, angiotensin II. These drugs, cause vasodilation and, therefore, a reduction in blood pressure.

22 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 Drugs may also act by inhibiting extracellular reactions. VIII. REGULATION OF ENZYME ACTIVITY The regulation of the reaction velocity of enzymes is essential if an organism is to coordinate its numerous metabolic processes. 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 23 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 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 VIII. REGULATION OF ENZYME ACTIVITY A. Regulation of allosteric enzymes Allosteric enzymes are regulated by molecules called effectors that bind noncovalently at a site other than the active site. 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 (Figure 5.16). Note: Allosteric enzymes frequently catalyze the committed step early in a pathway. 24 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 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. Dr. M. Alzaharna 2016

Dr. M. Alzaharna 2016 Figure 5.16: Effects of negative () or positive (+) effectors on an allosteric enzyme. A. Vmax is altered. B. The substrate concentration that gives halfmaximal velocity (K0.5) is altered. VIII. REGULATION OF ENZYME ACTIVITY A. Regulation of allosteric enzymes When the substrate itself serves as an effector, the effect is said to be homotropic. 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 exhibit cooperativity. These enzymes show a sigmoidal curve when reaction velocity (vo)

is plotted against substrate concentration ([S]), as shown in Figure 5.16. This contrasts with the hyperbolic curve characteristic of enzymes following Michaelis Menten kinetics, as previously discussed 26 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 1. Homotropic effectors: VIII. REGULATION OF ENZYME ACTIVITY A. Regulation of allosteric enzymes The effector may be different from the substrate, in which case the effect 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 (for example, 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 a re commonly encountered. For example, the glycolytic enzyme phosphofructokinase-1 is allosterically inhibited by citrate, which is not a substrate for the enzyme 27 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 2. Heterotropic effectors:

Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 Figure 5.17: Feedback inhibition of a metabolic pathway. VIII. REGULATION OF ENZYME ACTIVITY B. Regulation of enzymes by covalent modification Many enzymes are regulated by covalent modification, most often by the addition or removal of phosphate groups from specific serine, threonine, or 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 use ATP as the phosphate donor. Phosphate groups are cleaved from phosphorylated enzymes by the action

of phosphoprotein phosphatases (Figure 5.18). 29 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 Note: Protein phosphorylation is mediated by hormonal signals removal of phosphate groups. Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 Figure 5.18: Covalent modification by the addition and VIII. REGULATION OF ENZYME ACTIVITY B. Regulation of enzymes by covalent

modification 2. Response of enzyme to phosphorylation: Dr. M. Alzaharna 2016 Depending on the specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme. For example, phosphorylation of glycogen phosphorylase (an enzyme that degrades glycogen) increases activity, whereas phosphorylation of glycogen synthase (an enzyme that synthesizes glycogen) decreases activity 31 Dr. M. Alzaharna 2016 VIII. REGULATION OF ENZYME ACTIVITY C. Induction and repression of enzyme

synthesis The regulatory mechanisms described above 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. Enzymes subject to regulation of synthesis are often those 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 32 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016

The increase (induction) or decrease (repression) of enzyme synthesis leads to an alteration in the total population of active sites. VIII. REGULATION OF ENZYME ACTIVITY C. Induction and repression of enzyme synthesis In contrast, enzymes that are in constant use are usually not regulated by altering the rate of enzyme synthesis. Figure 5.19 summarizes the common ways that enzyme activity is regulated. 33 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016

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. Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 Figure 5.19: Mechanisms for regulating enzyme activity. [Note: Inhibition by pathway end product is also referred to as feedback inhibition.] IX. ENZYMES IN CLINICAL DIAGNOSIS Plasma enzymes 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 enzymes involved in blood coagulation.

These enzymes almost always function intracellularly and have no physiologic use in the 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 plasma. Increased plasma levels of these enzymes may indicate tissue damage (Figure 5.20). 35 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 Second, a large number of enzyme species are released from cells during normal cell turnover. or traumatized cells. Dr. M. Alzaharna 2016

Dr. M. Alzaharna 2016 Figure 5.20: Release of enzymes from normal and diseased IX. ENZYMES IN CLINICAL DIAGNOSIS A. Alteration of plasma enzyme levels in disease states Many diseases that cause tissue damage result in an increased release of intracellular enzymes into the plasma. The activities of many of these enzymes are routinely determined for diagnostic purposes in diseases of the heart, liver, skeletal muscle, and other tissues. Some enzymes show relatively high activity in only one or a few tissues. The presence of increased levels of these enzymes in plasma thus reflects damage to the corresponding tissue. For example, the enzyme alanine aminotransferase (ALT) is abundant in the liver. The appearance of elevated levels of ALT in plasma signals possible

damage to hepatic tissue. 37 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 B. Plasma enzymes as diagnostic tools IX. ENZYMES IN CLINICAL DIAGNOSIS

38 Isoenzymes (also called isozymes) are enzymes that catalyze the same reaction. However, they do not necessarily have the same physical properties because of genetically determined differences in amino acid sequence. Different organs commonly contain characteristic proportions of different isoenzymes. The pattern of isoenzymes found in the plasma may, therefore, serve as a means of identifying the site of tissue damage. For example, the plasma levels of creatine kinase (CK) are commonly determined in the diagnosis of myocardial infarction. Myocardial muscle is the only tissue that contains more than 5% of the total CK activity as the CK2 (MB) isoenzyme. Appearance of this hybrid isoenzyme in plasma is virtually specific for infarction of the myocardium. Dr. M. Alzaharna 2016

Dr. M. Alzaharna 2016 C. Isoenzymes and diseases of the heart X. CHAPTER SUMMARY Enzymes are not consumed during the reaction they catalyze. Enzyme molecules contain a special pocket or cleft called the active site. The active site contains amino acid side chains that participate in substrate binding and catalysis. The active site binds the substrate, forming an enzymesubstrate (ES ) complex. Binding is thought to cause a conformational change in the enzyme (induced fit) that allows catalysis. ES is converted to enzymeproduct (EP), which subsequently dissociates to enzyme and product. An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower free energy of activation. The enzyme does not change the free energies of the reactants or products and, therefore, does not change the equilibrium of the reaction. Most enzymes show Michaelis-Menten kinetics, and a plot of the initial reaction velocity (vo) against substrate concentration ([S]) has a hyperbolic shape similar to the

oxygen dissociation curve of myoglobin. 39 Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 Enzymes are protein catalysts that increase the velocity of a chemical reaction by lowering the energy of the transition state (Figure 5.23). 40 Any substance that can diminish the velocity of such enzyme catalyzed reactions is called an inhibitor. The two most commonly encountered types of reversible inhibition are competitive (which increases the apparent Km) and noncompetitive (which decreases the apparent Vmax). In contrast, the multisubunit allosteric enzymes frequently show a sigmoidal curve similar in shape to the oxygen dissociation curve of hemoglobin. The y typically catalyze the rate

limiting (slowest step) of a pathway. Allosteric enzymes are regulated by molecules called effectors that bind noncovalently at a site other than the active site. Effectors can be either positive (accelerate the enzyme catalyzed reaction) or negative (slow down the reaction). An allosteric effector can alter the affinity of the enzyme for its substrate, modify the maximal catalytic activity of the enzyme, or both. Enzymes can also be regulated by covalent modification and by changes in the rate of synthesis or degradation. Enzymes have diagnostic and therapeutic value in medicine. Dr. M. Alzaharna 2016 Dr. M. Alzaharna 2016 X. CHAPTER SUMMARY Dr. M. Alzaharna 2016 41

Dr. M. Alzaharna 2016

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