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usually ionic or hydrogen     * Usually results in a conformational change in the enzyme    * Lock and key model is outdated   * Induced Fit is used model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

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