Biochem Exam 2 (pt 2)

  1. Rennin or Rennet
    Enzymes produced by stomach of reminant mammals

    Some early canteens were made from animal stomachs; when milk was stored in these canteens, it clumped into cheese

    Chymosin is a protease enzyme that curdles the casein in milk
  2. Alcohol fermentation
    It is possible to convert glucose to ethanol via some action dependent on yeast
  3. Catalysts
    • Substances that increases the rate of a reaction 
    • Not consumed in the process
    • Do not affect thermodynamic equilibrium (they don't make the products more favorable, only accelerate the transition
  4. Bological catalysts
    • Most enzymes are proteins or protein-RNA complexes 
    • Ribozymes are RNA catalysts
  5. Enzymes are typically protein catalysts that exhibit:
    • Higher reaction rates- typically 106 to 1012 times faster than uncatalyzed reactions
    • Milder reaction conditions: below 100 degrees C. normal atmospheric pressure, and often near neutral pH
    • Greater reaction specificity
  6. Rate of reaction can be regulated by
    • Concentration of substrates and products 
    • or
    • Regulating enzyme activity via: allosteric control, covalent modification, variation of enzyme amount
  7. Active site
    The part of the enzyme which binds the substrate, and contains the residues that directly participate in making and breaking bonds
  8. Substrate
    A reactant, acted upon by the enzyme
  9. Inhibitor/Activator
    Substance that reduces or enhances the activity of an enzyme
  10. Cofactor/Coenzyme
    Small molecule required for enzyme activity (not for all enzymes)
  11. Holoenzyme
    Active enzyme bound to required cofactors
  12. Apoenzyme
    The enzyme without required cofactors
  13. Turnover
    One reaction cycle
  14. CAN Carbonic anhydrase
    • Catalyzes conversion of CO2 and H2O to carbonic acid
    • One of the fastest rxns
  15. Rate enhancement
    Factor by which reaction rates are enhanced by an enzyme= catalyzed rate/uncatalyzed rate
  16. Specificity in Enzyme-substrate interactions
    • Enzymes vary in their ability to distinguish between substrates
    • Some are very specific and only accelerates with one substrate
    • Most enzymes can act on a range of similar substrates i.e alcohol dehydrogenase (oxidizes small alcohols)
  17. 6 major classes of enzymes
    • Oxidoreductases
    • Transferases
    • Hydrolases
    • Lyases
    • Isomerases
    • Ligases
  18. Oxidoreductases
    Redox reactions
  19. Transferases
    Transfer of functional groups
  20. Hydrolases
    Hydrolysis reactions
  21. Lyases
    Group elimination to form double bonds
  22. Isomerases
    • isomerization reactions
    • converts from one isomer to another
  23. Ligases
    Bond formation coupled with ATP hydrolysis
  24. Kinases
    Transferases for a phosphate group
  25. Phosphatases
    Hydrolases for phosphoryl groups
  26. Proteases
    Hydrolases for amide bonds in proteins
  27. Thermodynamics vs Kinetics
    If a reaction has a negative change in Gibb's then you know a reaction can occur (favorable) but only kinetics tells you how quickly
  28. Activation energy
    Rate of reaction is defined by this which is required to transit a via a highly unstable transition state
  29. Image Upload 1
    What does this show?
    • The transition state is energetically unfavorable (high free energy) complex
    • The larger the change in energy of the transition state, the slower the reaction rate
    • -Transition state is unstable, therefore it is very small in the reaction mixture

    Transition state will quickly decompose to reactants or to products
  30. Rate-determining step
    • The slowest step: typically the formation of transition state
    • In a multi step reaction, the step with the largest activation energy is the rate limiting step
  31. Raising temp effect
    Reactions can be accelerated by temperature (T)

    Image Upload 2
  32. Lowering activation barrier (enzymes) effect
    • The lower the change of Gibbs at the transition state, more molecules have enough energy to cross the activation barrier
    • Enzymes provide a reaction pathway with a lower activation energy in order to reach equilibrium more quickly
    • Image Upload 3
  33. What is the shape of the curve for enzyme catalyzed reactions?
    Bell-shaped curve
  34. How can enzymes reduce gibbs free energy?
    They stabilize an intermediate state- compound state similar to the transition state
  35. Classes of rate enhancement exhibited by enzymes (mechanisms of catalysis)
    • Substrate entropy reduction: proximity, orientation
    • Preferential binding of the transition state
    • Acid-base catalysis
    • Covalent catalysis
    • Metal ion catalysis
  36. Entropy reduction: proximity, orientation
    • Enzyme-catalyzed rxns use standard organic mechanisms that are more efficiently than in solution
    • Proximity (local concentration) and orientation (proper alignment) are two obvious physical properties that can be manipulated by an enzyme

    Bringing reactants close together and with the enzyme catalytic center and aligning them for reaction speeds up reaction
  37. Outcome of proximity and orientation entropy reduction
    • Rate enhancement: up to 107-108
    • Large entropic penalty that must be compensated by binding energy of the enzyme-substrate complex
  38. Image Upload 4
    • Enzyme-substrate complex
    • Driven by free energy/affinity (inherent)
    • Energy barrier: solvation/desolvation and changes in structure/association (induced fit)
  39. Entropic penalty
    • Reflected in energy barrier between S and ES states
    • Image Upload 5 
    • First step is the formation of the enzyme-substrate complex (ES complex)
    • ES complex is typically more favored than free E and S due to enthalpic forces
    • EP complex is typically slightly less favored than free E and P to potentiate release of the product
  40. Transition state stabilization
    Energy difference for (Change Gcattransition state) for catalyzed reaction (EStransition-ES) is less than Change in G noncatalyzed transition state (Stransition-S) for a reaction that is enhanced by transition state stabilization
  41. Stickase: theoretical enzyme
    • Enzymes are not optimized for strong bonding to their substrates
    • Enzymes should (bind well) to transition states
    • Image Upload 6
  42. Enzyme-substrate complex
    • Multiple weak interactions between enzyme and substrate are responsible for ES complex
    • These weak interactions cover the "cost" of forming energetically unfavorable transition state

    Substrate is biased towards transition state in the bound enzyme-substrate complex (apply steric strain)
  43. How can transition state be mimicked by intermediate state
    Typically a covalent complex between enzyme and substrate
  44. What does increased affinity of the enzyme for the transition state relative to the substrate do?
    Increases the rate of the catalyzed reaction

    2 addtl H bonds between enzyme and transition state relative to substrate results in ~106 rate enhancement
  45. Acid-Base Catalysis
    Reactions can be catalyzed by proton transfer or sharing between a substrate and an enzyme (acid-base chemistry)

    Requires a side chain that can donate or accept a proton
  46. What is beneficial for amino acids serving as both an acid and base during different stages of the same catalytic cycle?
    Ensures the enzyme is returned to its original state at the end of the reaction allowing the enzyme to process another substrate
  47. pH optimum shape of Acid-Base catalysis
    • Typical dome-like shape
    • Optimal pH for most enzymes match their environment
  48. Covalent catalysis
    Enzymes may accelerate reaction rates through transient formation of enzyme-substrate covalent bonds

    Often involves reaction of a nucleophilic group with an electrophilic substrate (i.e sometimes called nucleophylic catalysis)
  49. Image Upload 7
    • Reaction coordinates of covalent catalysis
    • The more stable the intermediate, the harder it is to break the covalent bond

    The rate limiting step requires more energy to break
  50. Metal Ion Catalysis
    Metalloenzymes require a metal ion for catalytic activity
  51. Common metal ion catalysts
    • Fe2+
    • Fe3+
    • Cu2+
    • Mn2+
    • Co2+
    • Mo2+
  52. Structural metals
    • Na+
    • K+
    • Ca2+
  53. Structural or catalytic metals
    • Zn2+
    • Mg2+
  54. Major roles of functional metal centers
    • Binding to substrates to orient them for reactions
    • Oxidation-reduction reactions by changing the metal ion oxidation state
    • Electrostatic stabilization or shielding of negative charges

    Metal ions can make covalently bound water molecules more acidic than free water: good source of catalytic OH-
  55. Carbonic anhydrase: activation of water molecule
    Image Upload 8
  56. Alcohol dehydrogenase: stabilization of a transition state
    Image Upload 9
  57. Mechanism of catalysis for serine proteases
    • Proximity, orientation, and entropy reduction
    • Preferential binding of the transition state complex (transition state stabilization)
    • Acid-base catalysis
    • Covalent catalysis
  58. Common serine proteases
    • Chymotrypsin
    • Trypsin
    • Elastase
    • etc.
  59. Qualities of serine protease
    • The amide bond is very stable and difficult to break:
    • Standard temperature/environment: half-life ~20 years
    • Uncatalyzed: boil overnight in 6M HCl
    • Catalyzed: protease activity at physiological pH and temp continually breaks down proteins
  60. scissile bond
    covalent chemical bond that can be broken by an enzyme
  61. Substrate preference for chymotrypsin
    aromatic hydrophobic residues
  62. Substrate preference for trypsin
    Basic residues: Lys, Arg
  63. Substrate preference for elastase
    • Ala, Gly, and Val
    • but primarily Ala
  64. TEV protease
    • Catalytic domain of the Nuclear Inclusion-A protein from tobacco etch virus (TEV)
    • Image Upload 10
  65. Factor Xa

    • Coagulation cascade factors that enable blood clotting
    • Image Upload 11
  66. Enteropeptidase
    • Intestinal enzyme that cleaves inactive forms (zymogenes) of other proteolytic enzymes (e.g. chymotryspinogene, proelastase)
    • Image Upload 12
  67. Oxyanion hole
    • Filled by the tetrahedral intermediate, but cannot be accessed by the planar carbonyl of the amide bond
    • Amide protons from Gly193 and Ser195 make 2 additional hydrogen bonds to the tetrahedral transition state leading to significant stabilization of the of the transition state
  68. Zymogens
    Nonspecific proteases that are tightly controlled by their production in an inactive form that requires activation

    Enzymes produced in their inactive forms
  69. Digestive Zymogens
    Chymotrypsinogen, trypsinogen, and other zymogens are synthesized in the pancreas and secreted into small intestine

    Intestinal enteropeptidase cleaves trypsinogen to start an activation cascade
  70. Digestive zymogen clinical interest
    • Similar cascades are involved in blood coagulation
    • Deficiency in one of these coagulation factor proteases that disrupts the activation cascade causes hemophilia (poorly clotting blood leading to profound bleeding)
  71. Bovine Pancreatic Trypsin Inhibitor (BPTI) peptide
    • Competitive serine protease inhibitor
    • Forms stable complexes with and blocks the active sites of enzyme
    • Binding is reversible
  72. What does Bovine Pancreatic Trypsin Inhibitor (BPTI) inhibit
    • Trypsin, chymotrypsin, but also plasmin and plasminogen activator involved in thrombolysis
    • Used in surgeries as a drug (aprotinin) that inhibits thrombolysis

    Side effects: blood clotting (thrombosis)
  73. Kinetics
    • Study of reaction rates
    • Simplest of reactions: S(reactant) -> P (product)
  74. Velocity (v)
    • Reaction rate is equal to the disappearance of S and the formation of P over time (t)
    • Image Upload 13
  75. Enzyme-Substrate Complex equation
    • E+S -> ES complex -> EP complex -> E+P
    • With high [S], reaction is zero order with respect to S
    • Image Upload 14
  76. Simplification #1 of Michaelis-Menten Kinetics
    • Formation of product is the rate limiting step, therefore ES complex is formed promptly and EP ignored
    • Image Upload 15
  77. Simplification #2 of Michaelis-Menten Kinetics
    • If we only consider initial velocity (v0), at early time points P=0 and K-2=0
    • Image Upload 16
  78. Initial Velocity
    • moles/second product formation at reaction initiation
    • substrate is consumed as reaction proceeds, so [S] changes with time leading to an observed change in velocity
    • Image Upload 17
  79. Simplification #3 of Michaelis-Menten Kinetics
    If [S]>>[E]t, so the fraction of S that binds to E (to form ES) is negligible, and [S] is constant at early points
  80. Simplification #4 of Michaelis-Mentin Kinetics
    Steady-state assumption
    • Since k2 is the rate-limiting step (k-1>>k2), the formation and breakdown of ES quickly reaches equilibrium and [ES] is constant
    • v0=k2[ES]

    Image Upload 18
  81. Vmax
    When [S] is high, at saturation: [ES]~[E]tot, and maximum reaction velocity 

  82. Km (michaelis constant)
    • Image Upload 19
    • Equilibrium constant that has units of concentration
    • Km is the dissociation constant for the ES complex
    • Km is the substrate concentration ([S]) at which v0=vmax/2

    A large Km represents weak binding and a smaller Km represents stronger binding
  83. Under Michaelis-Menten conditions when [S]>>[E]
    • The steady-state assumption is in effect change in [ES]/change in t=0
    • k2 is the rate-limiting step and v0=k2[ES]
    • At saturation: [ES]~[E]Tot, and maximum reaction velocity vmax=k2[E]tot

    • Under these conditions:
    • Image Upload 20
  84. Lineweaver-Burk Plots
    Also referred to as Double-Reciprocal Plots

    • Use the Michaelis-Menten equation to form the line
    • Image Upload 21
  85. Basic lineweaver-burk plot
    Image Upload 22
  86. kcat
    • Catalytic constant: the turnover number or the number of reaction processes catalyzed per unit of time
    • kcat=vmax/[E]Tot

    Rxn is faster when kcat is higher
  87. What does kcat equal to under the Michaelis-Menten?
  88. Specificity constant
    • kcat/Km
    • A good measure of catalytic activity because it takes into account the rate of catalysis (kcat) and the enzyme-substrate interaction (Km)
  89. What does a larger specificity constant mean?
    Means the compound is a better substrate for the enzyme

    Represents the ability of an enzyme to convert substrate into product
  90. ACD toxin of Vibrio Cholerae
    • a non-michaelis-menten reaction
    • ACD covalently crosslinks actin into non-functional species
    • Also binds to one molecule of ATP and two molecules of actin
  91. Why do we care about inhibition?
    • Allosteric regulation (metabolism, signaling, etc.)
    • Drugs (e.g. antibiotics, anti-cancer drugs)

    Can be reversible and irreversible
  92. Types of reversible enzyme inhibition
    • Competitive
    • Uncompetitive
    • Noncompetitive
    • Mixed
  93. Competitive enzyme inhibition
    • The inhibitor competes with the substrate for binding to the enzyme active site
    • Transition state analogs are very strong competitive inhibitors
    • Image Upload 23
  94. Uncompetitive enzyme inhibition
    • Binds only after the substrate is bound
    • Works best when substrate concentration is high

    Image Upload 24
  95. Noncompetitive enzyme inhibition
    • Binds to an allosteric site, does not affect affinity for a substrate
    • Image Upload 25
  96. Mixed enzyme inhibition
    • Binds to an allosteric site, affects affinity for a substrate
    • Image Upload 26
  97. Irreversible enzyme inhibition
    Irreversibly modify a catalytically important active site residue

    Can be nonspecific and specific
  98. Targeted irreversible enzyme inhibition
    Inhibitors can be targeted towards reactive groups with specific activity: DIPF (diisopropyl phospho-fluoridate, DFP, DIFP) reacts specifically with serine residues

    In serine proteases, only one Ser is located in the active site and is sufficiently activated to react, other serine residues will not react
  99. Specific irreversible enzyme inhibition
    • Irreversible covalent modification can be combined with substrate recognition to generate a specific irreversible inhibitor
    • Also known as reactive substrate analogs
  100. Reversible enzyme inhibition
    • Reversible inhibitors bind to an enzyme, but can be removed
    • Each class effects enzyme kinetics differently
  101. Vmax in competitive inhibition
    Will not change since high substrate concentrations will eventually out-compete the inhibitor for binding

    • Inhibition is affected by the KI (the KD for the enzyme-inhibitor complex) and [I]
    • -Smaller KI= tighter binding= more inhibition
    • Image Upload 27
  102. Product inhibition
    • Where the product of the enzyme binds to the active site and down-regulates enzyme activity
    • A common form of competitive inhibition (negative feedback)
  103. Alpha in competitive inhibition
    The factor by which [S] must be increased to overcome the inhibitor
  104. Initial velocity equation in competitive inhibition
    Image Upload 28
  105. Competitive inhibition graphs
    Image Upload 29
  106. Lineweaver-Burk Plot for competitive inhibition including equation
    Image Upload 30
  107. Methanol poisoning
    Can lead to blindness and death but is treated with large amounts of ethanol

    The ethanol out-competes methanol for binding to alcohol dehydrogenase, preventing the conversion of methanol to toxic formaldehyde
  108. Image Upload 31
    Uncompetitive inhibition, the inhibitor binds directly (and exclusively) to the ES complex

    Inhibitor affects the catalytic function of the enzyme, but does not perturb substrate binding to the enzyme (S binding is improved)
  109. Initial velocity formula for uncompetitive inhibition and graph
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    • Image Upload 33
  110. Lineweaver-Burk Plot and equation for uncompetitive inhibition
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  111. Image Upload 35 Image Upload 36
    • Mixed and noncompetitive inhibition
    • The equilibrium distinguishes noncompetitive and mixed inhibition
  112. Mixed inhibition graph
    Special case where Km with inhibitor=Km without inhibitor is noncompetitive inhibition
  113. Lineweaver-Burk Plot for noncompetitive inhibition and equation
    Image Upload 37
Card Set
Biochem Exam 2 (pt 2)