Cell Biology: Enzymes

Properties of Catalysts

  • Accelerate chemical reactions without being changed themselves
    • Most catalysts are enzymes but some ribozymes (RNA)

Properties of Enzymes

  • Usually present in small quantities
  • Not irreversibly altered during reaction
  • Used many times
  • Enzymes can be saturated or unsaturated
  • May be extremely substrate specific
  • Accelerate a favorable reaction
  • Can be regulated
  • Have no effect on the thermodynamic properties of a reaction

​​Why Don’t Most Thermodynamically FavorableReactions Occur Quickly?

  • Most reactions require covalent bonds to be broken or formed
    • Requires certain amount of energy
  • Energy of Activation
    • Minimal energy of reacting molecules
    • Few molecules have the amount of energy required
  • Can increase the amount of reacting molecules by adding thermal energy
    • But is not practical in organisms
  • Must lower EA → by an enzyme (catalyst)

Enzymes and ΔG

  • Enzymes have no effect on ΔG
  • If there was no EA barrier, reactions would proceed unregulated

How Enzymes Work

  • Bring together reactants favorably
  • Accelerate bond breaking and bond forming
  • Enzyme-Substrate Complex
    • E+S ←→ ES ←→ E+P
  • Substrate interacts with active site 
    • Active site: groove or pocket on the surface of the enzyme
    • Made up of small clusters of amino acids that aren’t linear in the amino acid sequence
  • Accelerating Reactions
    • Orientation: needs to be in a specific orientation for reaction
    • Reactivity: charges (may be electron donation between active site and substrate
    • Inducing Strain: changing position of substrate to put strain on it and make it easier to break     

  Substrate Binding and Activation

  • Substrate bonding usually ionic or hydrogen
    • Usually results in a conformational change in the enzyme 
  • Lock and key model is outdated
  • Induced Fit is used model

  Enzyme Activity and Regulation

  • Dependent on factors 
    • pH 
    • Temperature
  • Can only perform duties in certain parts of the body where the pH and temperature is good for the enzyme 

  Catalytic Event

  • 1. Substrate bound and activated
  • 2. Temporary bonds formed between substrate and active site
  • 3. Covalent bonds in substrate adjusted
  • 4. Product released
  • 5. Cycle repeats 
  • Turnover number: number of time per second an enzyme can go through the entire sequence 
    • Specific for every enzyme 
      • And substrate
    • Varies widely depending on the requirements and needs of the cell
    • Max rate / enzyme concentration

  Enzyme Kinetics 

  • Graphs
    • Product vs time
  • Initial Reaction Velocity vs [Substrate]

Michaelis-Menten

  • \
    • K1- K4 are rate constants
  • Assumptions
    • Only looking at initial conditions
    • K4 << K3 → product does not rebind to enzyme
    • [ES] is nearly constant
    • [Etotal] = [E free] + [E bound]; [E bound] = [ES]
    • K1 >> K3 → formation of ES is much faster than breakdown of ES to E and P
  • Equation
    • K1 [E] [S] = K3 [ES] + K2 [ES]

       (rate of making) = (rate of breaking down)

  • K2 and K3 = 1/time

  • K1 = 1/time*concentration

  • V = Vmax [S] / Km + [S]

    • V = initial velocity of product formation
    • Rate of enzyme reaction, increases with increased substrate until Vmax
    • [S] = initial substrate concentration
    • Vmax = maximum possible reaction velocity
    • Enzymes working as fast as possible
    • Km = kinetic parameter (k2 + k3) / k1
    • Concentration of substrate at which rate is half Vmax

  • Vmax

    • Will occur when all enzyme is present in an ES complex
    • Velocity of product formation = k3 [ES]
    • Vmax = K3 [Etotal]
    • K3 = Kcat = Turnover Number

  • Km

    • At very low [S]
    • V = Vmax [S] / Km 
      • → [S] << Km so Km + [S] ~ Km
      • Ignoring [S] value
    • First order reaction with a linear slope (Vmax/Km)
    • Rate dependent on the [S]
    • Enzyme reaches Vmax at low [S] and enzyme binds tightly
    • At very high [S]
    • V = Vmax [S] / [S] → Vmax
      • → [S] >> Km so Km + [S] ~ [S]
      • Ignoring Km 
    • Zero order reaction (plateauing)
    • Vmax is initial velocity when [S] approaches infinity
    • Enzyme reaches Vmax at higher [S] and binds weakly 
    • When Km = [S]
    • V = Vmax [S] / 2[S] → Vmax/2
      • → [S] =  Km so Km + [S] ~ 2[S]

Roles of Km and Vmax

  • Km is independent of enzyme concentration; does not change when [E] changes
    • Consider it as playing for just one enzyme?
  • Km is specific for each enzyme catalyzed reaction
  • Vmax depends on the reaction conditions
    • Depends on enzyme concentration
    • More enzymes raise Vmax if the substrate stays the same?
  • Km and Enzyme Affinity for Substrate
    • High affinity: K1 = high, K2 = low
    • Low affinity: K1 = low, K2 = high
    • When K1 is smaller, Km is bigger 
    • Km is bigger → affinity is low
    • Km is smaller → enzyme affinity is high

Enzyme Inhibition

  • Irreversible
    • Caused by outside forces
    • Forms covalent bonds with enzyme
    • Causes loss of enzyme activity
    • Usually involves an amino acid at active site
    • Toxic to cells
  • Reversible
    • Binds to enzyme in a way that can be reversed
    • Active enzyme depends on inhibitor concentration of stability of Enzyme-inhibitor Complex
    • Competitive Inhibition
    • Mimics structure of the substrate
    • Binds to active site but cannot be made into product
    • Contests with the substrate for active site
    • Reduces enzyme activity 
    • Apparent change in Km → looks greater
      • Takes a higher concentration of substrate to meet  Vmax
      • Makes product slower -- need more substrate 
    • Substrate is fighting the inhibitor 
    • No change in Vmax
    • Makes enzyme less efficient
    • Noncompetitive Inhibition
    • Binds away from action site
    • Does not mimic substrate
    • Changes shape of active site so product cannot be formed
      • Substrate doesn’t fit into active site
    • Reduces enzymatic activity 
    • Enzyme is not functioning 
    • No relationship to concentration of substrate
      • Increasing substrate doesn’t help because it isn’t competing
    • Vmax is reduced because many enzymes unavailable 
      • Less enzymes to work with

 \n Regulation of Enzyme Activity 

  • pH

  • Compartmentalization: where in the cell the enzymes used

    • Ex: lysosomal enzymes made in lysosomes 
    • In acidic conditions as well

  • Inhibitors

  • Control concentration or availability of substrate

  • Allosteric regulation

  • Modification

  • Proteolytic Cleavage 

  • Substrate Level Regulation

    • Glucose + ATP → Glucose-6-Phosphate + ADP
    • Hexokinase used for reaction
    • Hexokinase is inhibited when there is a lot of product 

  • Allosteric Regulation

    • Regulatory molecules can cause enzymes to become active or inactive 
    • Can bind and cause inactivity or activity
    • PKA example
    • Changing shape of the enzyme to either have or not have an active site available 
    • Active: high affinity for substrate
    • Inactive: low affinity for substrate
    • Makes very little product

 

  • Feedback Inhibition
    • Product of one enzyme interacts with the enzyme that made it or another enzyme upstream in the pathway and inhibits enzyme activity
    • Version of allosteric and non competitive
    • Could affect any of these enzymes 
  • Regulation by Covalent Modification
    • Addition or removal of specific chemical groups
    • Phosphorylation
    • Phosphate group from ATP 
    • Usually activates but sometimes inactivates 
  • Proteolytic Cleavage
    • Enzymes start as inactive (precursors) when going into small intestine
    • Enteropeptidase comes in and chops some of the inactive enzyme off -- causing activation
    • Now activated trypsin turns on the other precursors by cutting off parts of the enzyme \n \n \n \n \n \n \n