Unit 3 1 UNIT 3 PART A: MYOGLOBIN PART B: HEMOGLOBIN PART C: INTRODUCTION TO ENZYMES PART D: ENZYME KINETICS PART A: MYOGLOBIN Asignment: Nelson & Cox, pp. 153 - 158 (stop at "Hemoglobin Subunits?"; skim pp. 155 - 159 section on "Protein-ligand Interactions Can Be Described Quantitatively"). Hemoglobin and myoglobin play vital roles in one of the most important aspects of animal metabolism - the utilization of oxygen. The most eficient energy - generating mechanisms in animal cels require molecular oxygen. Proteins that deliver oxygen to cels, and store it, until needed, are esential. Oxygen delivery is carefully regulated to met the demands of tisues. Studies on oxygen transporting proteins have provided vast insight into the function and regulation of proteins. Myoglobin stores and transports oxygen in vertebrate muscle tisue. The atomic structure of myoglobin was determined by x-ray crystalography in the 1950's. Because of the wealth of information on myoglobin it has become a clasic example of protein structure and function. Unit 3 2 1. Discuss the following terms: (pp. 153 - 154) a. Ligand b. Binding site c. Induced Fit d. Substrate e. Catalytic (active) site 2. Some important features of myoglobin (pp. 154 - 158): a. Why do multicelular organisms transport oxygen on Fe +2 incorporated into a heme group (p. 154)? b. Discuss the role of the heme group (pp. 154 - 155) 1) Identify the pyrrole ring 2) Point out the six coordination points to iron 3) Discuss the oxidation state of iron in myoglobin (and hemoglobin) c. What is a "globin" (p. 155)? Use Fig. 5-3 (p. 155) to describe the secondary and tertiary structure of myoglobin. Describe the location of the heme group in the protein. d. Based on what you learned in Unit 2, review how myoglobin folding occurs. Describe the polar or non-polar character of the inside and outside of the native protein. The oxygen binding site in myoglobin involves the heme group and two histidine residues. Is it surprising to find histidine residues in the interior of a protein? Explain. e. What is the physiological role of myoglobin (pp. 155, 159)? Unit 3 3 3. Use Fig. 5-4 (p. 156) to discuss the oxygen binding behavior of myoglobin. a. What is the meaning of P 50 (p. 158)? b. What is the P 50 of oxygen binding to myoglobin (Fig. 5-4, p. 156 [legend])? c. What does the hyperbolic shape of the binding curve tel you about the sensitivity of myoglobin to changes in ligand concentration (p. 159)? 4. Protein structure afects how ligands bind a. Use Fig 5-5 (p. 158) to discus the role of the distal histidine in the myoglobin ligand binding site. b. Discuss the role of the globin protein in preventing heme iron oxidation (p. 154). c. What is "molecular breathing" and why is it important in terms of myoglobin's function (p. 158)? Unit 3 4 PART B: HEMOGLOBIN Asignment: Nelson & Cox, pp. 158 - 170 (skip sections entitled "Cooperative Ligand Binding Can Be Described Quantitatively" and "Two Models Suggest Mechanisms for Cooperative Binding" on pp. 162 - 165). 1. Hemoglobin, in red blood cels, transports oxygen from the lungs to the tisues. a. Discuss the quaternary structure of hemoglobin (pp. 159 - 161): b. Use Fig. 5-6 (p. 159) to compare the 3° structure of a single subunit of hemoglobin to myoglobin. c. How similar is the 1° sequence of hemoglobin to that of myoglobin (Fig. 5-7, p. 159)? d. Where are the heme groups located in each subunit of hemoglobin? e. In the hemoglobin molecule, are the oxygen binding sites far apart or close together? f. What forces hold the subunits together? Use Fig. 5-9 (pp. 160) to discuss the salt bridges that stabilize the Tense conformation of deoxyhemoglobin. Use Fig. 5-10 (p. 161) to compare the conformations of the T and R states. g. Use Fig. 5-11 (p. 161) to ilustrate how binding of O 2 leads to conformational changes in the ligand binding site. How are these changes transmited to the subunit interface? h. Use Fig. 5-10 (p. 161) to compare the conformation of the hemoglobin T and R states. Unit 3 5 2. Hemoglobin binds oxygen cooperatively (pp. 161 - 162). a. Name the shape of the hemoglobin oxygen binding curve shown in Fig. 5-12 (p. 161). b. What is meant by the statement that the binding of O 2 by hemoglobin is cooperative? c. Do isolated hemoglobin subunits exhibit cooperativity in O 2 binding (p. 161)? Why or why not? d. Estimate the P 50 of oxygen binding for hemoglobin from Fig. 5-12 (p. 161)? Compare the P 50 for myoglobin to this estimate. Which protein has a higher afinity for oxygen? e. At physiologicaly relevant O 2 concentrations, which protein, myoglobin or hemoglobin, is more sensitive to smal changes in oxygen concentration (p. 159)? f. Hemoglobin is an example of an alosteric protein. Explain (p. 162). g. Diferentiate betwen the terms heterotropic and homotropic modulators. Which one is oxygen an example of? 3. Efects of Heterotropic Modulators on the Alosteric Behavior of Hemoglobin (pp. 165 - 167). a. The Bohr efect (p. 166) 1) In rapidly metabolizing muscle tisue much CO 2 is produced. In the red blood cel al the CO 2 forms H 2 CO 3 which ionizes to HCO 3 - + H + . In terms of hemoglobin binding to H + and O 2 , discuss how H + , produced in rapidly metabolizing tisues, promotes delivery of oxygen from the lungs to the tisues (p. 166). Unit 3 6 2) The mechanism of the Bohr efect: There are certain proton binding sites in hemoglobin that are of higher afinity in the deoxy form than in the oxy form. The increase in afinity for H + must reflect that the pKs of some groups are higher in deoxyhemoglobin than in oxyhemoglobin. What residues participate to give this Bohr efect (p. 166)? Explain why a closely positioned anion would increase the pK of histidine. b. Describe two ways in which CO 2 alostericaly lowers oxygen afinity (pp. 165 - 166). Be sure to consider the bicarbonate reaction in your response. c. Bisphosphoglycerate (BPG; pp. 167 - 168): 1) Use Fig. 5-17 (p. 167) to show the role BPG plays in influencing the oxygen afinity of hemoglobin? Describe the physiological significance of BPG. 2) Use Fig. 5-18 (p. 168) to show where in the deoxyhemoglobin structure the binding of BPG occurs? 3) What type(s) of interactions are involved in this binding (p. 167)? 4) Explain how this interaction afects hemoglobin's oxygen binding afinity. 5) Use Fig. 5-18 (p. 168) to explain what happens to the BPG binding site in oxygenated hemoglobin? NOTE: H + , CO 2 , and BPG al stabilize the T form of hemoglobin, thereby reducing its afinity for oxygen! Unit 3 7 4. For a review of myoglobin and hemoglobin structure and function, run through the 3-D Structure activity entitled " Oxygen-binding proteins " on the Lehninger Principles of Biochemistry 5th ed. website. These activities are browser specific. For details, read the notes on the external link from the Blackboard website (subsection coursework related links by unit, Unit 3). PART C: Introduction To Enzymes Asignment: Nelson & Cox, pp. 19 - 27, 183 - 192. A catalyst is a substance that increases the rate of a chemical reaction without itself being changed in the overal proces. Most biological catalysts are protein enzymes. If an enzyme is denatured or disociated into subunits, catalytic activity is usualy lost. Thus the primary, secondary, tertiary, and quaternary structures of protein enzymes are esential to their catalytic activity. 1. Distinguish betwen a coenzyme and a cofactor (p. 184). 2. Describe the key features of the active site of an enzyme (p. 186). 3. Distinguish betwen ?G° and ?G'° (p. 186). Unit 3 8 4. What determines the rate of a reaction (p. 187)? Refer to Fig. 6-2 (p. 186) in responding to the following questions: a. Is the standard fre energy of the reaction positive or negative? b. Is the equilibrium constant greater or les than 1? c. Does the equilibrium favor reactants or products? d. Does a favorable equilibrium ean that the rate of the forward reaction (reactants going to products) is fast? e. Does the presence of a catalyst have any efect on the position of the equilibrium? f. To undergo a chemical reaction, molecules must overcome a barier and be raised to a higher fre energy level. The fre energy barier betwen reactants and products represents the fre energy required for alignment of reactive groups, formation of transient structures, rearangement of bonds, and other transformations required for the reaction to occur in either direction. At the top of the fre energy hil is a point at which decay to products is optimal. This is the transition state. g. On the diagram, point out the standard fre energy of activation, ?G° ? , for both the forward and reverse reactions? h. Does a higher standard fre energy of activation correspond to a slower or faster reaction rate? i. Reaction rates can be increased by raising the temperature which increases the number of molecules with sufficient fre energy to overcome this fre energy barier. Is this temperature control a realistic variable in a biological system? j. Use Fig. 6-3 (p. 187) to show the afect of an enzyme on the standard fre energy of activation? Explain how this is acomplished. Identify the rate-limiting step for the enzyme-catalyzed reaction. Unit 3 9 5. Reaction equilibria are linked to ?G°, reaction rates are linked to ?G?. B R A k F k Consider the reaction above in which k F and k R are the rate constants of the forward and reverse reactions, respectively. Recal the following from your general chemistry: The rate of the forward reaction equals the rate constant, k F , times the actual concentration of A. The rate of the reverse reaction equals the rate constant, k R , times the actual concentration of B. At equilibrium the rates of the forward and reverse reactions are equal. Thus k F [A] = k R [B]. a. Write an expresion for Keq in terms of rate constants (p. 188). b. What is the efect of an enzyme on k F and k R ? on k F /k R ? c. What is the efect of an enzyme on the concentrations of reactants and products at equilibrium? 6. Using Fig. 6-6 (p. 191), explain that enzymes acelerate reactions by stabilizing the transition state. Unit 3 10 PART D: ENZYME KINETICS Asignment: Nelson & Cox, pp. 194 - 205 (skip pp. 200 ["Many Enzymes.."] - 201 ["Enzymes are Subject?"]). In this unit you wil learn some basic elements of enzyme kinetics. First you wil learn how simple kinetic measurements are made. Then you wil learn some of the basic tenets of what is now caled Michaelis-Menten kinetics. Many enzyme mechanisms are far more complex. Later you wil learn about alosteric enzymes. 1. Chemical kinetics Before analyzing the behavior of enzymes, let us review some chemical kinetics. a. For simplicity, consider an important ireversible "elementary" reaction, S ? P, the velocity (V) or rate of this reaction is the amount of P formed or the amount of S consumed per unit time, t. That is V = d [P] dt = d [S] dt Since this is an " elementary" reaction, the rate law is given directly from the stoichiometry of the reaction. V = k [S] The constant k is caled the rate constant for the reaction. In this case it is a "first order" rate constant because the rate is proportional to the first power of [S] and has units of sec -1 . Unit 3 11 b. In a reaction of the type A + B ? C, V = k [A] [B]. In this example the rate constant is "second order" and has units of M -1 sec -1 . (Note that the abbreviation M stands for molar, or moles per liter, whereas moles means an amount and not a concentration!) 2. Substrate concentration afects the rate of enzyme-catalyzed reactions. a. What is meant by V o ? Why is measuring V o considered to be a simplification (p. 194)? b. Use Fig. 6-10 (p. 194) to show how V o can be determined at a number of diferent [S]. Describe how this data is used to generate the Michaelis-Menton plot shown in Fig. 6-11 (p. 195). c. Define each of the following terms . Show how they can be determined on a plot of initial velocity vs [S] (Fig. 6-11, p. 195). 1) Km? 2) Vmax? c. The Michaelis-Menten Model 1) A crucial feature of the model is the existence of an ES complex. What aspect of the Michaelis-Menten plot (Fig. 6-11, p. 195) suggests the ES intermediate? 2) Discuss the Michaelis-Menten model (equation 6-10, p. 196) in terms of the following: a) The two fates of ES b) Rate constants c) Explain why the use of initial velocity (V o ) alowed Michaelis and Menten to ignore the reverse reaction (k -2 ) in their analysis of how V o varies with [S]. Unit 3 12 d. The Michaelis-Menten equation (equation 6-9, p. 196) 1) Study the Michaelis-Menten equation V = V [S] max K + [S] M o 2) Show that the equation fits the Michaelis-Menten plot by completing the folowing objectives. a) In what part of the Michaelis-Menten plot (Fig. 6-12, p. 197) is the velocity directly proportional to the substrate concentration? What wil be the form of the Michaelis-Menten equation at this part of the curve? b) In what part of the Michaelis-Menten plot (Fig. 6-12, p. 197) is the velocity independent of the substrate concentration? What wil be the form of the Michaelis-Menten equation at this part of the curve? c) Use the Michaelis-Menten equation to show that the velocity wil be half Vmax when [S] = Km. 3) Do Nelson & Cox problem 11 on page 230. e. The Lineweaver-Burk Double Reciprocal Plot (box 6-1, p. 197) 1) Describe how a Lineweaver-Burke plot can be generated from a Michaelis-Menten plot. 2) What shortcoming of the Michaelis-Menten plot is overcome in the Lineweaver-Burke plot? Unit 3 13 3) On Fig. 1 (Box 6-1, p. 197), show how the axis intercepts on a Lineweaver-Burke plot can be used to determine the Km and the Vmax of an enzyme. 4) Generate a double-reciprocal plot to complete Nelson & Cox problem 13 on page 231. f. Define the Michaelis constant, Km, in terms of thre rate constants (equation 6-24, p. 198). 1) In terms of rate constants, define the disociation constant K d for the following reaction where k 1 is the rate constant for the forward reaction, and k -1 is the rate constant for the reverse. What are the units of k 1 and k -1 ? k 1 E + S <=> E S k- 1 2) Under what conditions does K M = K d ? 3) Does a high value of K d or K m indicate a high or low afinity of E and S for each other? g. Define the turnover number (k cat ; p. 198). Do Nelson & Cox problem 15 on page 231. h. Catalytic perfection (p. 199) 1) What is the upper limit on k cat /K m ? 2) What does it mean if an enzyme has achieved catalytic perfection? i. Enzyme Inhibition (pp. 201 - 204) 1) Distinguish betwen reversible and ireversible inhibition. (p. 201; Note that aspirin is an example of an ireversible inhibitor because it acetylates the enzyme it inhibits.) Unit 3 14 2) Use Fig. 6-15 (p. 201) to distinguish competitive, uncompetitive, and mixed inhibition. For each type of inhibitor discuss the following: a) Can the enzyme simultaneously bind a substrate and an inhibitor? b) Can the efect of a given concentration of inhibitor be overcome by increasing the relative amount of substrate? 3) Inhibitors are kineticaly distinguishable. a) What is the efect of a competitive inhibitor on the apparent K m and apparent V max ? (Fig. 1, Box 6-2, p. 202) b) What is the efect of a uncompetitive inhibitor on the apparent K m and apparent V max ? (Fig 2, Box 6-2, p. 202) Note that the uncompetitive inhibitor lowers the apparent K m because it only inhibits at high substrate concentrations so that the substrate concentration giving half V max is lower. c) What is the efect of a mixed inhibitor on the apparent K m and apparent V max ? (Fig. 3, Box 6-2, p. 202) d) Note there is also a fourth type of inhibition, caled non- competitive. Inhibitors of this type change the apparent Vmax, but not apparent Km. e) Do Nelson & Cox problems 12 (pp. 230 - 231) and 19 (p. 232). f) What is a "suicide inactivator" (p. 204)? Jim Blankenship Microsoft Word - U03_F08.doc
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