drugs, diseases and enzymes

• what are the uses for enzymes?

  • diagnostics - enzyme levels or malfunction can indicate disease condition
  • therapeutic - they can be used to treat certain diseases
  • modes of inhibition
  • drug target - enzymes targeted with specific drugs to treat diseases. can combine enzymes with other things to make them more specific for targeting, for example to target a tumour.
• hormones can be derived by amino acids
• describe the first and second step in the biosynthesis pathway of adrenaline (amino acid derived hormone)?

  1. Adding an OH group onto the phenol ring of Tyrosine converts fit into L-DOPA
  2. Then the removal of CO2- turns L-DOPA into dopamine

  A) tyrosine hydroxylate

  B) DOPA decarboxylase

  
• describe the third and fourth step in the biosynthesis pathway of adrenaline?

  Third step: addition of another hydroxyl group produces noradrenaline Fourth step: substituting a H on the NH3+ with a methyl group produces adrenaline

  A) dopamine beta-hydroxylase

  B) Phenethanolamine N-methyltransferase

  
• if any enzyme on a pathway is inhibited/affected it can cause different diseases
• what is tyrosine hydroxyls deficiency?

  A rare metabolic disorder - this deficiency means the Tyrosine to L-DOPA conversion is affected and not enough L-DOPA is produced and so not enough Dopamine is produced
• what is dopamine?

  A neurotransmitter that can be naturally found in the brain where it enables neurone to communicate and control movement.
• how is tyrosine hydroxylase deficiency treated?

  Prescription of Sinemet (combination of Carbidopa and Levodopa)
• how does Sinemet treat TH deficiency/Parkinsons

  Levodopa (L-DOPA) can cross the blood brain barrier where it's converted to dopamine in the brain - catalysed by DOPA decarboxylate (DDC). DDC is also present on the body besides the brain so can do the conversion but dopamine is poor at crossing the BBB so not much will reach the target site. Carbidopa is a competitive inhibitor so inhibits the DDC and prevents the break down of L-DOPA outside the brain. This way L-DOPA stays in the circulation long enough to reach the brain and be converted into dopamine. Carbidopa can't pass the BBB.
• whats the advantage of using a competitive inhibitor with Levodopa?

  allows a lower dose of L-DOPA to be used which lowers side effects and higher bioavailability at the target site for more effective therapy
• what does the deficiency of dopamine beta-hydroxylase cause?

  causes the deficiency of norepinephrine (noradrenaline) and epinephrine (adrenaline) which causes symptoms like - dizzy head and difficulty standing
• how is the deficiency of dopamine beta-hydroxylasee treated?

  using droxidopa - which is a prodrug that can be converted directly to noradrenaline (norepinephrine) using the dopa-decarboxylate enzyme (which is also used to convert L-DOPA into dopamine)
• what is phenylketonuria?

  a genetic disease (inherited metabolic disorder) - when there's a lack of enzymes it can cause the accumulation of toxic levels of Phe (phenylalanine). those with this lack the enzyme phenylalanine hydroxylase

  
• how do you manage phenylketonuria?

  diet plan - low in phenylalanine. they need to avoid the artificial sweetener aspartame as if it hydrolyses it forms phenylalanine
• what are irreversible inhibitors?

  the inhibitor is hard to dissociate - is often a covalent modification in the active site

  e.g. penicillin binds transpeptidase + aspirin binds cycloxygenase
• what are reversible inhibitors?

  they form non-covalent interactions

  enzyme inhibitor complex forms but is easy to reverse

  3 modes of reversible inhibition are competitive, non-competitive and un-competitive

  e.g. methotrexate (anticancer) binds to dihydrofolate reductase
• what are the features of competitive inhibitors?

  • binds reversibly so has some structural resemblance to substrate - similar structure
  • inhibitor binds only to free enzyme
  • no reaction on inhibitor
  • more EI formation = less ES formation. Km(app) its increased
  • substrate blocked from, entering the active site so can be reversed by increasing [S]
  • inhibitor with high binding strength (low Ki) is more potent (powerful)
• what is Ki?

  the dissociation constant of the inhibitor - lower the Ki the slower the inhibitor will dissociate from the enzyme

  • measures how effective the inhibitor is 

  
• what are the features of non-competitive inhibitors?

  • can have both types; reversible for irreversible (covalently bonded)
  • inhibitor binds regardless at different site than substrate
  • binding to inhibitor interferes with normal conformational changes (induced fit) at active site required for catalysis - there won’t be an efficient reaction
  • can’t be reversed by increasing [S]
  • inhibitor with low Ki is more potent
  • if the inhibitor binds and causes the active site to not be exactly the shape you would normally get when induced fit, the Vmax is affected

  
• what the features of un-competitive inhibitors?

  • can have both types; reversible or irreversible (covalently bonded)
  • inhibitor binds at different site then substrate but only to ES. Site does not exist before ES is formed
  • binding to inhibitor interferes with normal conformational changes (induced fit) at active site required for catalysis
  • can’t be reversed by increasing [S]
  • inhibitor with low Ki is more potent

  
• how does competitive inhibitors affect the enzymes kinetic parameters?

  effect on Km - Km is raised as higher substrate concentration are required to achieve half of Vmax

  effect on Vmax - they don't effect this, it stays the same. the enzyme can still achieve its maximum catalytic activity
• how do non-competitive inhibitors affect enzyme kinetic parameters?

  on Km - Km stays the same because the inhibitor affects the enzyme-substrate binding independently of substrate concentration

  on Vmax - reduces Vmax because the inhibitor is binding to a site other than the active site and its present hinders the catalytic activity of the enzyme
• example of irreversible inhibitors?

  penicillin and aspirin
• how does penicillin inhibit transpeptidases?

  A OH group from the side chain of the serine residue of transpeptidase binds to the C7 of a penicillin (in the active site of transpeptidase) - this forms an inactivated penicillin-enzyme derivative.

  • so cell wall synthesis inhibited and bacteria can no longer replicate - no proper cross-linking = cell wall becomes weakened and more susceptible to osmotic pressure
• how does penicillin inhibit transpeptidases?

  A OH group from the side chain of the serine residue of transpeptidase binds to the C7 of a penicillin (in the active site of transpeptidase) - this forms an inactivated penicillin-enzyme derivative.

  So cell wall synthesis inhibited and bacteria can no longer replicate - no proper cross-linking = cell wall becomes weakened and more susceptible to osmotic pressure

  
• how does aspirin work as an irreverible inhibitor?

  Aspirin acetylates a serine residue in the active site of the COX enzyme (cyclooxygenase). The acylation of COX interferes with the binding of arachidonic acid to the active site. And so prostaglandins (what causes pain) is not produced and so nerve endings no longer stimulated so pain is killed.
• how does aspirin work as an irreverible inhibitor??

  Aspirin acetylates a serine residue in the active site of the COX enzyme (cyclooxygenase). The acylation of COX interferes with the binding of arachidonic acid to the active site. And so prostaglandins (what causes pain) is not produced and so nerve endings no longer stimulated so pain is killed.

  
• examples of reversible inhibitors?

  methotrexate and AZT
• how does methotrexate work as a reversible inhibitor?

  • methatrexate is a analog of folic acid and competitively inhibits dihydrofolate reductase (an enzyme that plays a role in producing of tetrahydrofolate, the active form of folic acid)
  • methatrexate binds to the enzyme x1000 fold higher
  • inhibits nucleotide base biosynthesis so used in cancer treatment
• what is tetrahydrofolate?

  a coenzyme for dihydrofolate reductase that makes purines and pyrimidines - DNA necessary for proliferation of cells
• how does AZT work as a reversible inhibitor?

  • azidothymidine is a potent inhibitor of HIV reverse transcriptase which catalyse the formation of viral DNA from viral RNA - this is needed for HIV replication
  • AZT competes with the substrate - thymidine