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Campbell6e lecture ch6

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Campbell6e lecture ch6

  1. 1. Chapter Six The Behavior of Proteins: Enzymes
  2. 2. Enzyme Catalysis <ul><li>Enzyme: a biological catalyst </li></ul><ul><ul><li>with the exception of some RNAs that catalyze their own splicing (Section 10.4), all enzymes are proteins </li></ul></ul><ul><ul><li>enzymes can increase the rate of a reaction by a factor of up to 10 20 over an uncatalyzed reaction </li></ul></ul><ul><ul><li>some enzymes are so specific that they catalyze the reaction of only one stereoisomer; others catalyze a family of similar reactions </li></ul></ul><ul><li>The rate of a reaction depends on its activation energy,  G° ‡ </li></ul><ul><ul><li>an enzyme provides an alternative pathway with a lower activation energy </li></ul></ul>
  3. 3. Enzyme Catalysis (Cont’d) <ul><li>For a reaction taking place at constant temperature and pressure, e.g., in the body </li></ul><ul><li>• the change in free energy is </li></ul><ul><li>Difference in energies between initial state and final state </li></ul><ul><li>The change in free energy is related to the equilibrium constant, K eq , for the reaction by </li></ul>
  4. 4. Enzyme Catalysis (Cont’d) <ul><li>Consider the reaction </li></ul>
  5. 5. Temperature dependence of catalysis <ul><li>• Temperature can also catalyze reaction (increase rate) </li></ul><ul><li>• This is dangerous, why? </li></ul><ul><li>• Increasing temperature will eventually lead to protein denaturation </li></ul>
  6. 6. Enzyme Kinetics <ul><li>For the reaction </li></ul><ul><ul><li>The rate of reaction is given by rate equation </li></ul></ul><ul><ul><li>Where k is a proportionality constant called the specific rate constant </li></ul></ul><ul><ul><li>Order of reaction : the sum of the exponents in the rate equation </li></ul></ul>
  7. 7. Enzyme Kinetics (Cont’d) <ul><ul><li>Consider the reaction </li></ul></ul><ul><ul><li>Whose rate equation is given by the expression </li></ul></ul><ul><ul><ul><li>Determined experimentally, not always from balanced equations </li></ul></ul></ul><ul><ul><li>The reaction is said to be first order in A , first order in B , and second order overall </li></ul></ul><ul><ul><li>Consider this reaction of glycogen with phosphate </li></ul></ul>
  8. 8. How Enzymes bind to Substrate <ul><li>In an enzyme-catalyzed reaction </li></ul><ul><ul><li>Substrate , S: a reactant </li></ul></ul><ul><ul><li>Active site : the small portion of the enzyme surface where the substrate(s) becomes bound by noncovalent forces, e.g., hydrogen bonding, electrostatic attractions, van der Waals attractions </li></ul></ul>
  9. 9. Binding Models <ul><li>Two models have been developed to describe formation of the enzyme-substrate complex </li></ul><ul><ul><li>Lock-and-key model : substrate binds to that portion of the enzyme with a complementary shape </li></ul></ul><ul><ul><li>Induced fit model: binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit </li></ul></ul>
  10. 10. 2 Modes of E-S Complex Formation
  11. 11. Formation of Product
  12. 12. An Example of Enzyme Catalysis <ul><li>Chymotrypsin catalyzes </li></ul><ul><ul><li>The selective hydrolysis of peptide bonds where the carboxyl is contributed by Phe and Tyr </li></ul></ul><ul><ul><li>It also catalyzes hydrolysis of the ester bonds </li></ul></ul>
  13. 13. An Example of Enzyme Catalysis (Cont’d)
  14. 14. Non-Allosteric Enzyme Behavior <ul><li>• Point at which the rate of reaction does not change, enzyme is saturated, maximum rate of reaction is reached </li></ul>
  15. 15. ATCase: An Example of Allosteric Behavior <ul><li>• Sigmoidal shape- characteristic of allosterism </li></ul><ul><li>• Again Max. velocity reached, but different mechanism </li></ul>
  16. 16. Michaelis-Menten Kinetics <ul><li>Initial rate of an enzyme-catalyzed reaction versus substrate concentration </li></ul>
  17. 17. Michaelis-Menten Model <ul><li>For an enzyme-catalyzed reaction </li></ul><ul><li>The rates of formation and breakdown of ES are given by these equations </li></ul><ul><li>At the steady state </li></ul>
  18. 18. Michaelis-Menten Model (Cont’d) <ul><li>When the steady state is reached, the concentration of free enzyme is the total less that bound in ES </li></ul><ul><li>Substituting for the concentration of free enzyme and collecting all rate constants in one term gives </li></ul><ul><li>Where K M is called the Michaelis constant </li></ul>
  19. 19. Michaelis-Menten Model (Cont’d) <ul><li>It is now possible to solve for the concentration of the enzyme-substrate complex, [ES] </li></ul><ul><li>Or alternatively </li></ul>
  20. 20. Michaelis-Menten Model (Cont’d) <ul><ul><li>In the initial stages, formation of product depends only on the rate of breakdown of ES </li></ul></ul><ul><ul><li>If substrate concentration is so large that the enzyme is saturated with substrate [ES] = [E] T </li></ul></ul><ul><ul><li>Substituting k 2 [E] T = V max into the top equation gives </li></ul></ul>
  21. 21. Michaelis-Menten Model (Cont’d) <ul><ul><li>When [S]= K M , the equation reduces to </li></ul></ul>
  22. 22. Linearizing The Michaelis-Menten Equation <ul><ul><li>It is difficult to determine V max experimentally </li></ul></ul><ul><ul><li>The equation for a hyperbola </li></ul></ul><ul><ul><li>Can be transformed into the equation for a straight line by taking the reciprocal of each side </li></ul></ul>
  23. 23. Lineweaver-Burk Plot <ul><ul><li>The Lineweaver-Burke plot has the form y = mx + b, and is the formula for a straight line </li></ul></ul><ul><ul><li>a plot of 1/V versus 1/[S] will give a straight line with slope of K M /V max and y intercept of 1/V max </li></ul></ul><ul><ul><li>such a plot is known as a Lineweaver-Burk double reciprocal plot </li></ul></ul>
  24. 24. Lineweaver-Burk Plot (Cont’d) <ul><ul><li>K M is the dissociation constant for ES; the greater the value of K M , the less tightly S is bound to E </li></ul></ul><ul><ul><li>V max is the turnover number </li></ul></ul>
  25. 25. Turnover Numbers <ul><ul><li>• V max is related to the turnover number of enzyme:also called k cat </li></ul></ul><ul><ul><li>• Number of moles of substrate that react to form product per mole of enzyme per unit of time </li></ul></ul>
  26. 26. Enzyme Inhibition <ul><li>Reversible inhibitor : a substance that binds to an enzyme to inhibit it, but can be released </li></ul><ul><ul><li>competitive inhibitor: binds to the active (catalytic) site and blocks access to it by substrate </li></ul></ul><ul><ul><li>noncompetitive inhibitor: binds to a site other than the active site; inhibits the enzyme by changing its conformation </li></ul></ul><ul><li>Irreversible inhibitor : a substance that causes inhibition that cannot be reversed </li></ul><ul><ul><li>usually involves formation or breaking of covalent bonds to or on the enzyme </li></ul></ul>
  27. 27. Competitive Inhibition <ul><ul><li>Substrate must compete with inhibitor for the active site; more substrate is required to reach a given reaction velocity </li></ul></ul><ul><ul><li>We can write a dissociation constant, K I for EI </li></ul></ul>
  28. 28. Competitive Inhibition
  29. 29. Competitive Inhibition <ul><li>In a Lineweaver-Burk double reciprocal plot of 1/V versus 1/[S], the slope (and the x intercept) changes but the y intercept does not change </li></ul>
  30. 30. A Lineweaver-Burke Plot for Competitive Inhibition
  31. 31. Noncompetitive Inhibition (Cont’d) <ul><li>Several equilibria are involved </li></ul><ul><li>The maximum velocity V max has the form </li></ul>
  32. 32. Noncompetitive Inhibition (Cont’d)
  33. 33. A Lineweaver-Burke Plot for Noncompetitive Inhibition <ul><ul><li>Because the inhibitor does not interfere with binding of substrate to the active site, K M is unchanged </li></ul></ul><ul><ul><li>Increasing substrate concentration cannot overcome noncompetitive inhibition </li></ul></ul>
  34. 34. A Lineweaver-Burke Plot for Noncompetitive Inhibition (Cont’d)
  35. 35. Other Types of Inhibition <ul><li>Uncompetitive - inhibitor can bind to the ES complex but not to free E. V max decreases and KM decreases. </li></ul><ul><li>Mixed - Similar to noncompetitively, but binding of I affects binding of S and vice versa. </li></ul>

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