enzymes

• Group specificity = any substrate with a particular functional group can bind to the active site
• Absolute specificity = only one specific substrate can bind
• Enzymes do not shift the equilibrium of the reaction, the equilibrium is just reached faster
• Energetics of catalysis: Gibbs free energy

  • A spontaneous reaction must have ∆G be negative
  • ∆G is independent of the path taken
  • At equilibrium the ∆G is zero
  • ∆G does not inform us about the rate of reaction
• What is an activated sugar nucleotide?

  An activated sugar nucleotide is a molecule that serves as a high-energy intermediate in various biochemical processes. These molecules play a crucial role in attaching sugar units to other molecules like proteins or lipids. The activation of a sugar nucleotide involves the addition of a carbohydrate attached to a nucleotide. The activated sugar nucleotide binds to enzymes' active sites.

  
  

  The energy released during the conversion of these activated forms to their respective products helps drive the otherwise energetically unfavorable reactions.
• What are the standard conditions for ∆G in bchem?

  • Reactants and products have concentrations of 1M, or 1 atm for gasses
  • Temperature of 298k
  • pH of 7- when H+ is a reactant, it has a concentration of 1M
• Enzymes decrease the activation energy (G‡) to overcome the activation energy barrier.

  • enzymes lower the activation energy by stabilizing the transition state (X‡)
• Where does the energy come from to lower the activation energy?

  Binding energy:

  • free energy known as binding energy is released when many weak interactions occur between the enzyme and substrate
  • Only the correct substrate can maximize the amount of binding energy, thus increasing the specificity
  • maximal binding energy occurs during the transition state
• Features of the enzyme active site:

  1. Residues forming the active site come from different positions in the sequence (proteins far apart in the sequence may actually be folded in a way that makes them physically close in the active site)
  2. Makes up a small volume of the total protein
  3. Unique microenvironment designed to interact with the substrate (could be non-polar or polar to bind to specific substrates)
  4. Forms multiple weak interactions with substrates
  5. Specificity of substrate binding depends on precise arrangement of active site atoms (and the atom's identity)
• Enzymes' active site often appears as a cleft that allows interactions with the substrate
• What are cofactors?

  Cofactors are non-protein compounds that bind to proteins and are needed for biological activity. Cofactors include:

  • Co-enzymes: vitamin-derived organic molecules
  • Metals (Zn2+, MG2+, Ni2+, etc.)

  Apoenzyme= enzyme without the cofactor

  Holoenzyme = enzyme with the cofactor
• Prosthetic group: cofactor binds very tightly to active site (helpers) - you'd rarely find enzymes without them

  Cosubstrate: loosely bound cofactor that binds to and is released from the enzyme with each reaction
• What is the lock and key model of enzyme-substrate binding?

  Perfect fit between enzymes' active site and substrate binding site
• What is the induced fit model?

  Enzyme is dynamic and atoms in active site are rearranged upon binding of substrate to become complementary after binding.
• What is the first order reaction equation for A→P?

  V = k[A]
• What are the units for the first order rate constant, k?


  s-1
• What is the reaction order equation for 2A→P or A + B→P?

  Second order reactions:

  V= k[A]^2 or V=k[A][B]
• What are the units for the second order rate constant?

  M-1 s-1
• what is a pseudo first order reaction?

  V=k[A][B]

  • If [B]>>[A] and A is present at low concentrations then the reaction will not appear to depend on [B]
• Reactions can be zero order and therefore not depend on the concentration of reactants
• General equation for enzymatic reaction

  A) Enzyme substrate complex

  B) Can be ignored since very little will go back to ES

  
• Michaelis-Menten kinetics

  A) Km

  B) Vmax

  C) K-1

  
• The Michaelis-Menten model assumes steady-state kinetics

  • Pre-steady state: Initial mixing of E + S, while [ES] builds up
  • Steady-state: [ES] remains approximately constant (i.e., the rate of ES formation = rate of ES breakdown)
  • Steady-state kinetics: Measurements of initial velocity while [ES] is relatively stable
• What happens when [S]<<Km?

  [S] in the denominator can be ignored, and velocity is directly proportional to [S]

  
• What happens when [S]>>Km?

  Ignore Km, and the reaction has a zero order reaction rate (ie, independent of [S].

  
• What happens if [S]=km?

  Km must be equal to the [S] where the reaction proceeds at half the rate of its maximal velocity.

  A) [S] + [S]

  B) 2

  
• Km is an important enzyme characteristic


  Depends on the:

  • Substrate
  • pH
  • Temperature
  • Ionic strength

  Does not depend on: enzyme concentration

  • Km is the concentration of substrate at which half of the enzyme molecules are bound to substrate
  • The cellular concentration of a particular substrate is often close to km, why might this be beneficial?
  • at Km, enzymes are already at a significant velocity and sensitive to changes in [S]
• How to determine Km and Vmax using a Lineweaver-Burk plot.

  A) y

  B) x

  C) slope

  D) Vmax

  E) Vo

  F) Vmax

  G) -1/km

  H) Km/Vmax

  
• The turnover number

  The turnover number (Kcat) is the number of substrate molecules that the enzyme can turn into product per unit time when fully saturated with enzyme.

  A) Vmax/[E]t

  
• 1/Kcat = time for a single reaction
• Catalytic efficiency

  Kcat/Km is a measure of catalytic efficiency and is known as the specificity constant. It takes into account the rate of catalysis (kcat) and the nature of the enzyme substrate interaction (Km).

  When [S]<<Km then:

  Vo = Kcat/Km [S] [E]t

  • kcat/km is the rate constant for ES formation
• Kcat/km is always less than K 1
• K1 is the rate of formation of the enzyme substrate complex and the rate limiting step. The only limit on K1 is how fast enzyme can encounter substrate in solution (diffusion)
• Temperature and enzyme catalysis

  Enzyme activity increases with temp until enzyme is denatured as the backbone cannot handle the increased movement and unfolds
• Allosteric enzymes regulate the flux of biochemicals through metabolic pathways: they catalyze the committed step in a pathway.

  • committed step = first step to make the product (b/c once you start, you can’t go back)
• How can we control the amount of final product “F” to get just the right amount?

  Allosteric enzymes have more than one active site, allowing them to bind to both stimulatory and inhibitory molecules at regulatory sites (sites on the enzyme other than the active site).

  • Inhibitory molecules bind to the regulatory site (eg. final products) to inhibit reaction
  • Stimulatory molecules (eg. a metabolite early in the pathway) bind to the regulatory site, increasing enzyme activity. Process known as feed forward activation / stimulation.
• Allosteric enzymes do not follow michaelis-menten kinetics and display sigmoidal kinetics.

  • Two models for explaining the sigmoidal kinetics of an allosteric enzymes
  • Concerted
  • Sequential
• Concerted model for allosteric enzymes:

  • Relaxed form (R) catalyzes reactions
  • Tense form (T) is less active but more stable and common
  • R and T are in equilibrium
  • L0 is the allosteric constant. L0= T/R, and L0 is typically in the hundreds since T form is more common
  • Symmetry rule: all active sites of an individual enzyme must be in the same state
  • Substrate binds more readily in the R form
  • The binding of substrate shifts the T and R equilibrium in favour of R when substrate binds to active in R, trapping all other active sites on enzyme in R form
  • Behaviour known as cooperativity
• Threshold effect: allosteric enzymes are more sensitive to changes in [S] near Km than Michealis-Menten enzymes with the same Vmax. (ie, less substrate is needed to reach Vmax)
• What are the units of the Michaelis-Menton constant Km?

  Concentration of substrate