MCB 102 Lec 7 Enzymes and Kinetics

  1. What are enzymes?
  2. What do catalysts do?
    Increase rxn rates without being used up
  3. What do most enzymes look like?
    Globular proteins
  4. What are some other things that catalyze reactions?
    Some ribozymes and ribosomal RNA
  5. What is the oldest field of biochemistry, dating back to late 1700s?
    Study of enzymatic processes
  6. What does enzyme literally mean?
    In yeast
  7. How did enzyme get its name?
    Eduard Buchner discovered in 1897 that yeast extracts could ferment sugar to alcohol
  8. What did Buchner's findings with fermentation show?
    Fermentation was promoted by molecules that continued to function when removed from cells
  9. What do enzymes need when they can't catalyze reactions by themselves?
    • Cofactors
    • Coenzymes
    • Cofactors AND coenzymes
  10. What is a cofactor?
    One or more inorganic ions
  11. What are some examples of cofactors?
    • Fe2+
    • Zn2+
    • Mn2+
  12. What is a coenzyme?
    • Complex organic or metallorganic molecules
    • Usually they act as transient carriers of specific functional groups
  13. What is a prosthetic group?
    An enzyme that is commonly found with its cofactor or coenzyme tightly bound
  14. What are some examples of prosthetic groups?
    • Holoenzyme
    • Apoenzyme/Apoprotein
  15. What is a holoenzyme?
    Enzyme + prosthetic group
  16. What is an apoenzyme/apoprotein?
    The protein part of a holoenzyme
  17. What are the different classifications of enzymes based on their functions?
    • Oxidoreductases
    • Transferases
    • Hydrolases
    • Lyases
    • Isomerases
    • Ligases
  18. What type of reaction is catalyzed by oxidoreductases?
    Transfer of electrons (hydride ions or H atoms)
  19. What type of reaction is catalyzed by transferases?
    Group transfer rxns
  20. What type of reaction is catalyzed by hydrolases?
    Hydrolysis rxns (transfer of functional groups to water)
  21. What type of reaction is catalyzed by lyases?
    Cleavage of C-C, C-O, C-N, or other bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds
  22. What type of reaction is catalyzed by isomerases?
    Transfer of groups within molecules to yield isomeric forms
  23. What type of reaction is catalyzed by ligases?
    Formation of C-C, C-O, and C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor
  24. What does the rate of any reaction depend on?
    • Concentration of reactants
    • Rate constant (k)
  25. What is the rate/velocity of a reaction denoted by?
  26. What is the equation for the rate/velocity of a first order reaction?
    V = k[S]
  27. How many substrates are involved in a first order reaction?
  28. What is the equation for the rate/velocity of a second order reaction?
    V = k[S1][S2]
  29. How many substrates are involved in a second order reaction?
  30. What does the apostrophe on Keq and G° mean?
    At pH 7
  31. What does the rate constant (k) depend on?
    • Several factors
    • Especially free energy (ΔG)
  32. How do enzymes affect the rate constant (k)?
    Lower the free energy (ΔG)
  33. What is the kappa in the rate constant equation?
    Bolzmann constant
  34. What does the h stand for in the rate constant equation?
    Planck's constant
  35. What is the relationship between the rate constant and the activation energy of a reaction?
    Inverse and exponential
  36. Free energy, G, is plotted as a function of what?
    Reaction progress (reaction coordinate)
  37. What is the starting point of a forward or reverse reaction called?
    Ground state
  38. What is ground state?
    The starting point for a forward or reverse reaction
  39. The free energy change that occurs during a reaction under a standard set of conditions is denoted as what?
  40. What is ΔG°?
    The free energy change that occurs during a reaction under a standard set of conditions
  41. What are the standard set of conditions that free energy change occurs under?
    • T = 298 K
    • P = 1 atm
    • C = 1 M solute concentrations
  42. What is ΔG'°?
    Standard free-energy change at pH 7
  43. What is the change in Gibbs free energy relationship?
    ΔG = ΔH - TΔS
  44. How do enzymes affect reaction rates?
    By lowering the activation energy of reactions
  45. What is a transition state?
    A "fleeting molecular moment" in which events have equal likelihood of transitioning to substrate or product
  46. If enzymes can't change the direction of a reaction, what can they do?
    Can catalyze it in either direction depending on the G
  47. Do enzymes affect the equilibrium of a reaction?
  48. What does the ΔG curve for a catalyzed reaction look like?
    Multiple little peaks going from S → ES → EP → P
  49. What does the ΔG curve for a normal reaction look like?
    One tall peak
  50. What are the hallmarks of enzyme-mediated catalysis?
    • Tremendous rate enhancement
    • Occurs under mild (physiological) conditions
    • Precise reaction
    • Stereo-specific products formed in high yield
    • Subject to metabolic regulation
  51. What are the mild physiological conditions under which enzyme-mediated catalysis occurs?
    • pH 7
    • 37° C
    • Aqueous soln
    • Low concentration of the reactants
  52. How do enzymes accelerate reactions?
    By facilitating approach to and formation of the transition state
  53. Although enzymes accelerate reaction rate, they do not change the _______ of the reaction.
  54. How do enzymes lower the activation energy?
    • Covalent bonds
    • Non-covalent interactions
  55. How do enzymes lower the activation energy through covalent bonds?
    • Functional groups on enzymes can react with groups in the substrates
    • Form transient covalent bonds
    • Lower energy path for the reaction
  56. How do enzymes lower the activation energy through non-covalent interactions?
    • The substrate and enzyme form an ES complex that is stabilized by non-covalent interactions
    • This releases energy known as binding energy, Gb that is used to lower the energy barrier
  57. What is binding energy used for?
    Lower the energy barrier
  58. What is the ES complex stabilized by?
    Non-covalent interactions
  59. What kind of energy is released when non-covalent interactions are formed between the enzyme and substrate?
    Binding energy
  60. Through what process does binding contribute to catalysis?
    • Entropy reduction
    • Substrate desolvation
    • Induced fit
  61. Catalysis of an enzyme requires the formation of what?
    An Enzyme-Substrate complex
  62. Where does a substrate bind on an enzyme?
    • The active site
    • Like a pocket
  63. How does a higher activation energy affect the rate of a rxn?
    • Lower rate constant
    • Slower rxn
  64. How does an active site expedite a chemical rxn?
    • 1. Entropy gain from substrates shedding water of solvation and displacing water bound in the active site
    • 2. Molecular complementary- geometry of the pocket "fits" the substrates and maximizes van der Waals contacts
    • 3. H-bonds and salt bridges between groups on the substrates and side chains in the active site are stronger in the absence of competition from or shielding by water
    • 4. Binding at the active site holds the substrates in close proximity (raises their effective local concentration)
    • 5. Binding at the active site holds the substrates in a precise orientation (promotes productive encounter)
    • 6. Some binding energy used to introduce strain or polarization into the bonds in the substrates
  65. How do the H-bonds and salt bridges in enzyme acitve sites affect the transition state?
    • Release binding energy by forming bonds/interactions
    • Makes energy available
    • Used to lower transition state
  66. How does the precise orientation of substrates in an enzyme active site expedite a reaction?
    • Enzymes tend to put substrates in comformations where they could react with each other
    • Promotes productive encounter
    • Not random
    • Conformation favors reaction
  67. How does the close proximity of substrates in an active site of an enzyme expedite a reaction?
    Raises their effective local concentration
  68. What does it mean for the binding energy to be used to introduce strain or polarization into the bonds in the substrates?
    • Active site is most complementary to the reactants in their transition state
    • Both the enzyme and its substrates change- "induced fit"
  69. Why must enzymes be complementary to the reaction transition state to catalyze a reaction?
    • Without the enzyme, a substrate (like a metal stick for example) will go into a transition state (bent) and then produce products (broken stick)
    • If the enzyme only fit the substrate, it wouldn't have room to go into transition state (bend) and wouldn't produce the products
    • With the enzyme most complementary to transition state, it provides room for the transition state and is most effective
  70. Energetically, how is it better to have enzymes be complementary to the transition state?
    • The increase in free energy needed to reach the transition state is partially offset (or paid for) by the interaction between the enzyme and the substrate in the transition state (binding energy)
    • Pretty much, the binding energy given off by the interaction between enzyme and transitioning substrate makes up for whatever free energy (G) would normally be needed to reach the transition state
  71. How do enzymes achieve their catalytic activity?
    Weak binding interactions between the enzyme and the substrate provide a substantial driving force for enzymatic catalysis
  72. How do enzymes achieve their specificity?
    Derived from the formation of many weak interactions between the enzyme and its specific substrate
  73. What is enzyme kinetics?
    Refers to the rate of a rxn and how it changes in response to experimental parameters
  74. How are most enzyme/substrate reactions written as?
    E + S ⇌ ES ⇌ E + P
  75. What is the intermediate step in an enzyme/substrate rxn?
    ES (duh)
  76. Which part of the enzyme/substrate rxn is slower?
    ES ⇌ E + P
  77. Which part of the enzyme/substrate rxn is negligible, and why?
    • k-2
    • That side of the rxn is slower so it's not likely it'll go in that direction
  78. What did Michaelis and Menton propose about the enzyme/substrate reaction?
    • The first part of the rxn is very fast, leading to the formation of the ES complex
    • The second step, which releases the enzyme and product was proposed to be slower
    • Since this second reaction is slower, the [ES] is considered the limiting factor in the rxn and it accumulates as the reaction progresses
  79. What's usually in excess in the enzyme/substrate rxn?
    The substrate
  80. What is the pre-steady state?
    • When we first mix the enzyme with excess substrate
    • There's a moment when the concentration of ES is constantly increasing
    • Lasts only milliseconds
  81. What is a steady state?
    • After the pre-steady state
    • When the concentration of ES becomes constant
    • Enzyme saturates
  82. When we measure Vo, what realm are we working in?
    Steady state kinetics
  83. Vo varies with what?
    Substrate concentration
  84. What does the speed of a rxn depend on?
    Affinity of enzyme for substrate
  85. What is Vmax?
    • Enzyme is saturated
    • Can't go any faster, no matter hwo much substrate there is
  86. What is the Michaelis-Menten Equation?
  87. What is the definition of Km?
    the concentration of substrate at which the enzyme is operating at half its maximal velocity
  88. What does Km represent?
    • The affinity of the enzyme for the substrate
    • How easy it is to form ES complex and for ES complex to dissociate
    • Larger Km = dissociating quicker, lower affinity
  89. What's the equation for Km?
  90. What is the equation for Vmax?
    Vmax = k2[E]total
  91. What is Vo a measure of?
    • Initial velocity
    • How quickly/well the enzyme catalyzes rxn
  92. When does Km = [S]?
    When  chart?chf=bg,s,00000000&cht=tx&chl=V_o%20%3D%20%5Cfrac%7BV_%7Bmax%7D%7D%7B2%7D&chs=158x70
  93. What is the Lineweaver-Burk Plot?
    Linearized, Double-Reciprocal
  94. What is the equation for chart?chf=bg,s,00000000&cht=tx&chl=%5Cfrac%7B1%7D%7BV_o%7D&chs=40x70?
  95. What is the slope of the Lineweaver-Burk Plot?
  96. What is chart?chf=bg,s,00000000&cht=tx&chl=%5Cfrac%7B1%7D%7B%5BS%5D%7D&chs=40x78?
    The x axis
  97. What is chart?chf=bg,s,00000000&cht=tx&chl=%5Cfrac%7B1%7D%7BV_%7Bmax%7D%7D&chs=78x70?
    • The y intercept
    • chart?chf=bg,s,00000000&cht=tx&chl=%5Cfrac%7B1%7D%7BV_o%7D&chs=40x70 when [S] is very large
  98. What is chart?chf=bg,s,00000000&cht=tx&chl=-%5Cfrac%7B1%7D%7BK_m%7D&chs=82x70?
    chart?chf=bg,s,00000000&cht=tx&chl=%5Cfrac%7B1%7D%7B%5BS%5D%7D&chs=40x78 when Vo is very large
  99. What would you do to determine Km from a double-reciprocal plot?
    Multiply the reciprocal of the x axis intercept by -1
  100. What is kcat/Km a measure of?
    Catalytic/enzymatic efficiency
  101. Define the catalytic constant (kcat) of an enzyme.
    • The limit rate of any enzyme-catalyzed reaction at saturation
    • Measure of how fast you are in your enzymatic capabilities
    • How quickly you convert ES complex into product
    • Also called turnover number
  102. What is the limit rate of the rxn we've been working with (E + S ⇌ ES → E + P)?
    kcat = k2
  103. What does the turnover number change this equation into? chart?chf=bg,s,00000000&cht=tx&chl=V_o%20%3D%20%5Cfrac%7Bk_2%5BE_1%5D%5BS%5D%7D%7BK_m%20%2B%20%5BS%5D%7D&chs=208x86
  104. Enzyme efficiency is limited by what?
  105. What does high Km mean?
    • EM dissociates quicker
    • Lower affinity
  106. What does low Km mean?
    • EM dissociates slower
    • Higher affinity
  107. Most desirable enzyme has what kind of kcat/Km ratio?
    • Low Km
    • High kcat
    • Bigger ratio number, the more efficient
  108. How can an enzyme gain efficiency?
    • Having high velocity
    • Having high affinity
  109. Which is a more efficient enzyme: catalase vs. acetylcholinesterase?
    • Well...
    • Catalase has a faster kcat rate
    • But acetylcholinesterase has a higher affinity
    • BUT overall, the kcat/Km ratio is bigger for acetylcholinesterase... so AChE is the winner
  110. What are inhibitors?
    • Compounds that decrease enzyme's activity
    • Molecules that somehow slow down or stop/prevent rxn
    • Can be reversible or irreversible
  111. What are irreversible inhibitors?
    • Inactivators
    • React with the enzyme
    • One inhibitor molecule can permanently shut off one enzyme molecule
    • Often powerful toxins but also may be used as drugs
  112. What are reversible inhibitors?
    • Often structural analogs of substrates or products
    • Often used as drugs to slow down a specific enzyme
  113. What can reversible inhibitors bind to?
    • The free enzyme and prevent the binding of the substrate
    • The enzyme-substrate complex and prevent the reaction
  114. What are the different kinds of reversible inhibitors?
    • Competitive
    • Uncompetitive
    • Mixed
    • Non-competitive (subset of mixed)
  115. What is competitive inhibition?
    Inhibitor competes with substrate for binding
  116. What do competitive inhibitors do?
    • Binds to free enzyme at active site
    • Will not affect Vmax of rxn; but [S] at which you need Vmax will
    • Does not affect catalysis
    • Apparent increase in Km
  117. What is "apparent Km"?
    • The Km measured with a competitive inhibitor
    • Gonna be higher because it'll take more substrate to get it to 1/2 Vmax
  118. What does the Lineweaver-Burk plot look like with a competitive inhibitor?
    • Lines intersect y-axis
    • chart?chf=bg,s,00000000&cht=tx&chl=-%5Cfrac%7B1%7D%7BK_m%7D&chs=82x70 is smaller
  119. If you have like 10 substrates and 2 enzymes, and those enzymes are taken up by inhibitors, what must you do to get the same results from the rxn?
    Add a lot more substrate to outcompete the inhibitors
  120. What do uncompetitive inhibitors do?
    • Binds to [ES]
    • Does not affect substrate binding
    • Similar to having less enzymes, b/c occupied by inhibitor so you can't use it
    • Inhibits catalytic function
    • Less enzyme, decrease in Vmax; available enzymes will saturate sooner
    • Apparent decrease in Km
    • No change in chart?chf=bg,s,00000000&cht=tx&chl=%5Cfrac%7BK_m%7D%7BV_%7Bmax%7D%7D&chs=78x78
  121. What does the Lineweaver-Burk plot look like with an uncompetitive inhibitor?
    • Lines are parallel
    • Shifts to the left
  122. At high concentrations of S, Vo approximates what, instead of the usual, with an uncompetitive inhibitor?
    Approximates chart?chf=bg,s,00000000&cht=tx&chl=%5Cfrac%7BV_%7Bmax%7D%7D%7B%5Calpha'%7D&chs=78x70 instead of the usual Vmax
  123. What happens to Vmax as more inhibitor is added?
    Vmax decreases
  124. What does Vmax depend on?
    Amount of enzymes
  125. What about the Vmax equation can be changed? What can't?
    • k2 can't be changed; enzymatic property
    • Can change [ES]; either E or S
  126. How can you increase Vmax?
    • Changing [S] won't do anything cuz there's already a bunch of substrate
    • Vmax was showing that enzyme was saturated (all enzymes occupied)
    • Adding [E] will increase Vmax
  127. What is mixed inhibition?
    • Binds enzyme with or without substrate
    • Inhibitor binds to [ES]
    • Also binds to [E]
    • Binds at regulator site
    • Inhibits both substrate bindinb and catalysis
    • Leads to complicated changes
    • Decrease in Vmax
    • Apparent change in Km
  128. What does the Lineweaver-Burk plot look like with mixed inhibition?
    Lines intersect at the left of the y-axis
  129. What is non-competitive inhibition?
    • Subset of mixed inhibition
    • Does not compete with the substrate for the binding site
    • Binds at a separate site
    • Decreae in Vmax (you have less enzyme available)
    • No change in Km
    • When rate for binding (k) of [ES] = k binding [E]
  130. What does the Lineweaver-Burk plot look like with non-competitive inhibition?
    • Lines intersect at the x-axis
    • Increase in chart?chf=bg,s,00000000&cht=tx&chl=%5Cfrac%7B1%7D%7BV_%7Bmax%7D%7D&chs=78x70
  131. Enzyme activity regulation can be:
    • non-covalent modification
    • covalent modification
    • irreversible
    • reversible
  132. What does allosteric mean?
    Showing the alteration of the activity of a protein through binding of an effectro molecule at a specific site
  133. The kinetics of what kind of modulators differ from Michaelis-Menten kinetics?
  134. What are allosteric regulators?
    • Modulators
    • Bind at different site; not the active site
    • Still affects rate of function of enzyme
    • Affects affinity of substrate for enzyme (and vice versa)
    • Doesn't affect Vmax
  135. In what way do allosteric modulators affect the affinity of enzymes?
    • Can modify it in a positive or a negative way
    • Can increase or decrease affinity
  136. What is allosteric inhibition?
    Inhibitor binding to another location; not the active site
  137. What is ubiquitination?
    • Adding ubiquitin molecule to enzyme
    • The more added, the more enzyme is degraded
  138. What's an example of ADP-ribosylation?
    How cholera bacteria affects function of some enzymes or proteins
  139. What are some reversible covalent modifications?
    • Phosphorylation
    • Adenylylation
    • Acetylation
    • Myristorylation
    • Ubiquitination
    • ADP-ribosylation
    • Methylation
  140. What are zymogens?
    • Non-active enzymes
    • Most enzymes travel as zymogens throughout the body
Card Set
MCB 102 Lec 7 Enzymes and Kinetics
MCB 102 Lec 7 Enzymes and Kinetics