Macromolecules

Calmodulin Calmodulin Hemoglobin Lysozyme

Proteins: Enzymes

Table of Contents

  1. Enzyme Attributes
  2. Thermodynamics
  3. Spontaneity
  4. Active Sites
  5. Enzyme Kinetic Properties
  6. Plotting Enzymatic Reactions
  7. Enzyme Inhibition

Enzyme Attributes
  1. Enzymes optimize the orientation of their substrates, ensuring maximum reactivity between molecules
  2. Enzymes are efficient and highly specific
  3. Enzymes are highly regulated
    1. Feedback inhibition
    2. Regulatory molecules
      1. enhance
      2. inhibit
    3. Covalent modification
      1. acetylation
      2. methylation
    4. Proteolytic activation (zymogen: an inactive precursor)
      1. cleaved from an active protein

Thermodynamics

First Law of Thermodynamics

  1. Total energy of systems and surroundings is constant

Second Law of Thermodynamics

  1. A reaction is spontaneous only if the sum of the entropies of the system and the surroundings increases
* Change in S -> degree of disorder increases for a spontaneous reaction
* Total energy [Change in E depends on initial and final states only: independent of the pathway]
* Free energy equation [Delta G = Delta H(enthalpy) - T Delta S(entropy)]
* Heat content [Delta H = Delta E + P Delta V]

Spontaneity
  1. A reaction is spontaneous only if the free energy is negative
    1. Catabolic reactions
  2. When the free energy is zero the system is in equilibrium
  3. Energy is necessary if Delta G is positive
  4. Delta G depends on Greactants - Gproducts
    1. Free energy is independent of the pathway
  5. Enzymes alter the pathway from reactants to products by changing the activation energy
    1. Enzymes do not alter the final equilibrium of the reaction, they just hasten it
  6. Catalyzed reactions depend on an enzyme substrate complex
    1. E + S <-> ES <-> E + P (enzyme and product)
    2. The substrate occupies the enzyme's active site

Active Sites
  1. Small in comparison to the whole enzyme
  2. Three dimential structure: amino acids far apart in the 1o structure can be close in 3o structure
  3. Substrates bind through weak interactions (additive)
  4. Contain clefts and crevices
  5. Binding of substrate depends directly on atomic arangement

Enzyme Kinetic Properties
* Most enzymes follow Michalis - Menten kinetics (>95%)
  1. If the [S] is small then the velocity of the reaction is dependent on the [S]
  2. If the [S] is large then the velocity of the reaction is independent of the [S]
  3. Km = Michalis Menten constant
    1. Km is a measure of the affinity that an enzyme has for a given substrate
    2. A high Km = a low affinity
    3. A low Km = a high affinity
    4. Km = [S] at 1/2 Vmax
    5. V = (Vmax)([S]/[S] + Km)

Plotting Enzymatic Reactions
  1. We can use linear replots of enzymatic data to obtain Km and Vmax
  2. This is done by measuring the velocity of a reaction at varying [S]

Types of plots

  1. Lineweaver - Burke plot (Double - Reciprocal plot)
    1. X-axis = 1 / [S]
    2. X-intercept = -1 / Km
    3. Y-axis = 1 / V
    4. Y-intercept = 1 / Vmax
    5. Slope = Km / Vmax

  2. Hanes - Wolf linear plot
    1. X-axis = [S]
    2. X-intercept = -Km
    3. Y-axis = [S] / V
    4. Y-intercept = Km / Vmax
    5. Slope = 1 / Vmax

  3. Eadie - Hofstee plot
    1. X-axis = V/[S]
    2. X-intercept = Vmax / Km
    3. Y-axis = V
    4. Y-intercept = Vmax
    5. Slope = -Km

  4. Eisenthal - Cornish - Bowdin linear replot
* a plot of V and -[S]
* Experiments are rerely neat an converge on the same point
* Bad data will show up easily
  1. X-axis = [S]
  2. X-intercept =
  3. Y-axis = V
  4. Y-intercept =
  5. Slope =

Enzyme Inhibition
Reversible types of inhibition
  1. Competitive
    1. Substrate and inhibitor both compete for the same enzyme active site
    2. The proble can be overcome by large amounts of substrate
    3. High [S] will outcompete the I for the active site
  2. Non-competitive
    1. Vmax appears to decrease with an increasing [I]
  3. Un-competitive
    1. Rare
    2. A series of parallel lines affecting both Km and Vmax
      1. Vmax decreases
      2. Km decreases

* It is possible to which type of inhibition is occuring by examining the pattern on a Lineweaver - Burke plot (LB plot)