Enzyme inhibition (competitive, non-competitive and uncompetitive)

In the presence of a noncompetitive inhibitor, Vmax is decreased, whereas Km is unchanged. The steady-state concentration of ES is decreased.

Substances that reduce the activity of an enzyme are called inhibitors.

Reversible inhibitors bind to an enzyme but rapidly dissociate from it [in contrast to irreversible inhibitors , which bind tightly and dissociate very slowly from the enzyme].

There are several types of reversible inhibitors:
 

Ans. is ‘d’ i.e., Is a competitive antagonist of acetylcholine

o Atropine acts as competitive antagonist at muscarinic receptors. It has no activity on nicotinic receptors and has nothing to do with Ach release.

Ans. is ‘b’ i.e., Acetazolamide

o Acetazolamide is non-competitive , reversible inhibitor of carbonic anhydrase.

A i.e. Competitive inhibitor of acetylcholine; C i.e. Hypothermia prolongs the block

Non-Depolarizing Neuromuscular Block

Non-depolariszing muscle relaxants are competitive blockers of ACh receptors on NAM. They don’t cause depolarization.

Among non-depolarizing blockers only, mivacuronium is metabolized by pseudocholineateraseQ. – Block is reversed by neostigmine and other anticholinesterases.

Block is potentiated by hypokalemia and Mg⁺² Q

Ca⁺² increases the release of ACh from nerve endings;thus partially antagonizes the blockQ Acodosis increases the duration and degree of block.

Mild cooling antagonize the block but greater cooling (<33°C) potentiates block Q

Ans. is ‘a’ i.e., Imatinib and suntinib

Molecular targeted agents

• Tyrosine kinase inhibitors
• Competitive inhibitors → Imatinib, Nilotinib, Sunitinib, Dasatinib, Erlotinib, Gefitinib, Lapitinib, Sorafinib (Remember all ends with ‘ nib’).
• Monoclonal antibodies → Cetuximab, panitumomab.
• HER₂/neu (ERB B₂) inhibitors Monoclonal antibody – Transtuzumab.
• Targeted antibody → Gemtuzomab (anti CD-33), Rituximab (anti – CD20), Alemtuzumab (anti CD-52).
• Vascular endothelial growth factor (VEGF) inhibitor → Monoclonal antibody – Bevacizumab.
• Proteosome inhibitors → Bortezomib.
• Histone deacylase inhibitor → Vorinostat
• DNA – methyl transferase inhibitor → 5-azacytidine, 2-deoxy-5 azacytidine.
• All – trans – retinoic acid.
• Biological response modifier – Recombinant IL-2 (aldesleukin, denileukin).

Ans. is ‘b’ i.e., Non competitive

ENZYMATIC ACTIONS

• Drugs can either increase or decrease the rate of enzymatically mediated action. A.

Stimulation

• Stimulation of enzymes by drugs, that are truly foreign substances, is unusual.
• Enzyme stimulation is relevant to many endogenous mediators and modulators, eg: adrenaline stimulates adenylyl cyclase.
• Stimulation of an enzyme increases its affinity for the substrate so that rate constant (Km) of the reaction decreases.
• Enzyme activity can also be increased by enzyme induction ie synthesis of more enzyme. In this Km does not change.

Ans. is ‘a’ i.e., Potency reduced

Competitive antagonist

1. Antagonist bind to the same receptors as agonist
2. Antagonist resembles chemically with the agonist
3. The same maximal response can be obtained
4. Potency is reduced (Right shift of DRC)
5. Km is increased but Vmax is unchanged

Non competitive antagonist

1. Binds to another site of receptor
2. Does not resemble
3. Maximal response is supressed
4. Efficacy is reduced (Flattening of DRC)
5. Km is unchanged but Vmax. is reduced.

Ans: B.)Non-competitive inhibition

In the graph shown in the image, X-axis shows the Velocity(rate of reaction), Y-axis shows the substrate concentration,”A’-shows normal enzyme,”B” shows competitive inhibition and “C” shows non-competitive inhibition.

• This maximum rate of reaction is characteristic of a particular enzyme at a particular concentration and is known as the maximum velocity or Vmax.
• The substrate concentration that gives you a rate that is halfway to Vmax is called Km.
• Competitive inhibitors:
  • They impair reaction progress by binding to an enzyme, often at the active site, and preventing the real substrate from binding.
  • At any given time, only the competitive inhibitor or the substrate can be bound to the enzyme (not both).
  • Competitive inhibition acts by decreasing the number of enzyme molecules available to bind the substrate.
  • With a competitive inhibitor, the reaction can eventually reach its normal Vmax
  • but it takes a higher concentration of substrate to get it there.Vmax is unchanged but Km is higher
• Noncompetitive inhibitors:
  • They don’t prevent the substrate from binding to the enzyme.
  • In fact, the inhibitor and substrate don’t affect one another’s binding to the enzyme at all.
  • However, when the inhibitor is bound, the enzyme cannot catalyze its reaction to produce a product. 
  • Thus, noncompetitive inhibition acts by reducing the number of functional enzyme molecules that can carry out a reaction.
  • With a noncompetitive inhibitor, the reaction can never reach its normal Vmax regardless of how much substrate we add.Km is unchanged.

Ans. is ‘d’ i.e., Non-competitive irreversible inhibition

T^ypes of enzyme inhibition

• Enzyme inhibition is of two types :‑
• Reversible and
• Irreversible
• Reversible inhibition

Reversible inhibition may be of following types :‑

i) Competitive inhibition (substrate analogue inhibition)

• The inhibitor competes with substrate for catalytic (substrate-binding) site of enzyme.
• Inhibitor resembles substrate in structural configuration.
• Inhibition can be reversed by increasing the concentration of substrate.
• Competitive inhibition increases km of the enzyme but Vmax does not change.
• Examples of competitive inhibition are inhibition of succinate dehydrogenase by malonate, HMG CoA reductase by statins, carbonic anhydrase by acetazolamide and LDH by oxamate.

ii) Non-competitive inhibition

• Inhibitor binds at a site other than the substrate-binding site.
• Thus inhibitor can bind to both free enzyme and enzyme substrate complex.
• The inhibitor therefore lowers the concentration of active enzyme.
• A non-competitive inhibitor cause decrease in Vmax but no change in Km.
• Inhibition can be reversed only by exhaustive dialysis of inhibited enzyme.

Ans. is ‘a’ i.e., In competitive inhibition Vmax unchanged but Km increased 

Type of inhibitor             Km                         Vmax

Reversible inhibbitor

Competitive                      Increased                 No effect

Non-competitive               No effect                  Decreased

Uncompititive                   Decreased                Decreased

Irrversible inhibitor           No effect                  Decreased (same as reversible competitive)

Ans. is ‘d’ i.e., Non-competitive irreversible inhibition 

Ans. is ‘b’ i.e., Non-competitive inhibitor

Non-competitive inhibitor

• In this type of inhibition no competition occurs between substrate and inhibitor.
• Inhibitor is structurally different from substrate and binds to enzyme at a site other than the substrate binding (catalytic) site.
• Occupancy of this site by the inhibitor alters the shape of the enzyme such that its catalytic activity is reduced or lost.
• The substrate is still able to bind the enzyme, but the enzyme cannot catalyze the reaction when inhibitor is bound. 
• Thus, the non-competitive inhibitor does not block the active site of enzyme, but behaves as though it were removing active enzyme from the solution.
• Evidently, unlike competitive inhibition, increasing the substrate concentration does not reverse the inhibition. 
• Non-competitive inhibition may be reversible or irreversible.

Ans. is ‘b’ i.e., Acetazolamide

• Carbonic anhydrase inhibitors act by a non-competitive, reversible inhibition of the enzyme carbonic anhydrase.

Carbonic anhydrase inhibitors (acetazolamide)

• Carbonic anhydrase (CAse) is an enzyme which catalyzes the reversible reaction H₂0 + CO₂ <—> H₂CO, H 1-1++HCO3-.
• The enzyme is present in renal tubular cells (especially PT), gastric mucosa, exocrine pancrease, ciliary body of eye, brain and RBC.
• Carbonic anhydrase inhibitors inhibit intracellular CAse in PT cells resulting in slowing of hydration of CO₂ —> decreased availability of H⁺ to exchange with luminal Na⁺ through Na⁺-1-1⁺ antiport.
• Inhibition of brush border CAse retards dehydration of H₂CO₃ in the tubular fluid so that less CO2 diffuses back into the cells.
• The net effect is inhibition of HCO3- (and accompanying Na^t) reabsorption in PT –> alkaline diuresis. Secretion of fr in DT and CD is also inhibited. Though Fr is secreted at this site by Fr-ATPase, it is generated in
• the cell by CAse mediated reaction. As such, this is a subsidiary site of action of CAse inhibitors.
• For the same degree of natriuresis CAse inhibitors cause most marked kaliuresis compared to other diuretics.
• Read below
• Collecting tubule is the most important site of K.’ secretion by the kidney.
• IC⁺ secretion occurs in exchange with Ne.
• If Na’ load is higher in collecting tube’s it will be absorbed in exchange with K^± and more K⁺ will be secreted, i.e. higher the Na⁺ load in CD, higher will be K⁺ excretion in urine.
• Carbonic anhydrase inhibitors act on proximal tubules, where maximum Na+ (70%) is absorbed —) they block this absorption and will cause maximum kaliuresis (K⁺ excretion in urine).

Extrarenal actions of acetazolamide

• Lowering of IOT due to decreased formation of aqueous humour.
• Decreased gastric HCL and pancreatic NaHCO₃ secretion.
• Raised level of CO₂ in brain and lowering of pH  sedation and elevation of seizure threshold.
• Alteration of CO₂ trasport in lungs and tissues.

Uses of CAse inhibitors

• Glaucoma               
• Acute mountain sickness                                
• Periodic paralysis
• Epilepsy                     
• To alkalinise urine for UTI or to promote excretion of acidic drugs
• Dorazolamide and brinzolamide are used topically for glaucoma.
• Adverse effects
• Hypersensitivity reactions in patients sensitive to sulfonamides (CAse inhibitors are sulfonamide derivatives).
• Bone marrow depression, metabolic acidosis, hypokalemia, drowsiness, paresthesias, fatigue and abdominal discomfort.
• In hepatic diseases may precipitate hepatic coma by interfering with urinary elimination of NH3. 
• Contraindications for carbonic anhydrase inhibitors.
• COPD  
• Hepatic cirrhosis           
• Hyperchloremic acidosis
• Carbonic anhydrase inhibitor (acetazolamide) can cause allergic reactions in patients hypersensitive to sulfonamides.