Enzymes

• Irreversible inhibitors: covalently bind enzymes
• Reversible inhibitors: non-covalently associate with enzymes can have

  • Competitive Inhibition
  • Uncompetitive Inhibition
  • Mixed iNhibition / non-competitive
• Competitive Inhibition resembles the substrate and compete for binding to the enzyme active site
• Competitive Inhibition increases apparent Km. Reach Vmax/2 faster. When linear, all inhibitors intersect Y-axis at 1/Vmax
• Uncompetitive Inhibition is where inhibitor binds to enzyme substrate complex at site distinct from substrate binding site and prevents reaction from occuring. Affects Vmax and apparent Km
• Uncompetitive inhibition decreases apparent Km and Vmax. When linear different concentrations of I are parallel and do not intersect.
• Mixed inhibition can have inhibitor bind to site distinct of substrate binding site but can bind to the enzyme or the enzyme substrate complex to prevent a reaction from occuring
• Mixed inhibitors decreases Vmax but Km can be decreased or increased.
• Noncompetitive is when α=α'. Vmax is reduced and Km is unaffected
• For mixed inhibitors:

  • if α>α' then it intersects before above the X axis
  • If α<α' then lines intersect below the x axis
  • If α=α' then they intersect on the x axis
• Allosteric enzyme has effector bind to regulatory site and substrate bind to active site
• When allostery is in effect:

  • Enzymes don't obey Michaelis-Menten Kinetics
  • There is a sigmoidal activity curve with cooperative substrate binding with two states High or low activity
  • Often associated with feedback inhibition
• Effectors also known as modulator:

  • Bind non-covalently to regulatory sites- heterotropic or homotropic
  • Can increase activity (positive) or decrease (negative) enzyme activity
• Homotropic or homoallostery influences state to change to R and substrates act as effectors. They can bind active or regulatory sites
• Heterotropic or Heterallostery has non-substrate effectors so different molecules that bind to regulatory sites
• Effectors can increase or decrease K0.5

  • If it is a positive heterotropic activator it decreases K0.5
  • Positive heterotropic inhibitor increases K0.5
• Symmetry model of allostery

  • Quaternary structure
  • Each oligomer exists as R or T state
  • Ligands or substrates bind to both states with different affinities shifting equilibriums
  • Overall symmetry is maintained so all subunits are in the same conformation
• Sequential Model of Allostery:

  • proteins have quaternary structures
  • Each subunit exists as R or T
  • Overall protein symmetry is not maintained as all subunits do not have to be same conformation
• Aspartate Transcarbamoylase or ATCase

  • Transferase
  • Quaternary structure with 6 catalytic and 6 regulatory subunits
  • D3 symmetry
  • Feedback inhibited by CTP
  • Activated by ATP
  • In R state has no steric conflicts and free rotation
  • In T state two catalytic monomers have steric conflict
• Feedback inhibition: the concentration of end product of pathway signals pathway when to stop. Many have allosteric modulators
• PALA binds to stabilize R state because it tricks it thinking it looks like ATCase so molecule stays in R state
• CTP is a heterotropic inhibitor while ATP is heterotropic activator and aspartate is homotropic activator. When ATP and CTP is present you get activation as ATP binds to a higher affinity.
• Phosphofructokinase is a:

  • phosphotransferase molecule
  • D2 symmetry / homotetramer
  • Each subunit has active site and regulatory site.
• Reversible Covalent Modification:

  • Phosphorylation to Ser, Thr, Tyr, His
  • Adenylation- With Tyr add AMP to enzyme
  • ADP-Ribosylation- uses NAD+
  • Palmitoylation- lipid anchor
• Protein Kinase generally phosphorylates substrates by modification of specific residues within target sequence. The target sequence acts as a substrate for the kinase.
• Protein Kinase A regulates glycogen phosphorylase by phosphorylation:

  • Phosphorylated phosphorylase kinase is active compared to unphosphorylated version that is less active
  • The active phosphorylase kinase then can activate glycogen phosphorylase a to be more active
  • ATP phosphorylates along with the kinase
• Glycogen synthase catalyzes glycogen synthesis through multiple phosphorylation sites at mostly C termini. When phosphorylated it inhibits the enzyme and lowers activity but doesn't fully shut off
• Because glycogen synthase has multiple phosphorylation sites:

  • each site can be affected by different kinases
  • Each site is associated with reduction in activity
  • Can have multiple phosphorylations simultaneously to reduce activity
• Concerted regulation: the same protein has two different targets and it turns one on and one off. Effective for coordinated regulation
• Isocitrate Dehydrogenase in E. Coli:

  • Inactive when phosphorylated because it blocks substrate binding and has negative charge repelling citrate
  • Acts as an on and off switch
• Irreversible covalent modifications:

  • are changes in polypeptide primary structure like cutting
  • Occur in zymogens to activate enzymes
  • When enzymes need to be destroyed or inactivated
• Digestive enzyme tract:

  1. Trypsinogen is an inactive zymogen that is activated by a peptidase to form trypsin the active proteinase
  2. Trypsin is feedback inhibitor and activates chymotrypsin the inactive zymogen to pie-chymotrypsin the active form
  3. pie-chymotripsin can activate itself to undergo covalent change to form α-chymotripsin the fully mature active proteinase
  4. α-chymotripsin feedback inhibits pie from creating more
• Trypsin cleaves Chymotrypsinogen between R and I residue while pie chymotrypsin is cleaved between Y and T residues