Enzyme inhibition (competitive, non-competitive and uncompetitive)

INTRODUCTION:
  • The chemical substances (organic or inorganic) which interfere with enzyme activity are called as inhibitors (negative modifier)the process is called as enzyme inhibition. 
TYPES:
  • 3 broad categories: (based on recovery after removal of inhibitor)
  1. Reversible inhibition
  2. Irreversible inhibition
  3. Allosteric inhibition
REVERSIBLE INHIBITION:
  • Combine non-covalently with the enzyme
  • Rapid dissociation of EI complex
  • Can be readily removed by dialysis
  • Fully active Enzyme can be recovered after removal of Inhibitor.
Reversible inhibitions are further classified as:
  1. Competitive inhibition
  2. Non-competitive inhibition
  3. Un-competitive inhibition
COMPETITIVE INHIBITOR:
  • When the active site or catalytic site of an enzyme is occupied by a substance other than the substrate of that enzyme, its activity is inhibited. The type of inhibition of this kind is known as competitive inhibition.
  • In such inhibition both the ES and EI (Enzyme-Inhibitor) complexes are formed during the reaction.
  • However, the actual amounts of ES and EI will depend on:
  1. Affinity between enzyme and substrate/inhibitor,
  2. Actual concentrations (amounts) of substrate and inhibitor present, and
  3. Time of preincubation of enzyme with the substrate or inhibitor.
  • With the increase in conc. of inhibitor lowers the rate of enzymatic reaction.
  • Thus, the Km is high, but Vmax is the same in competitive inhibition.
  • However, when the concentration of substrate is increased, the effect of inhibitor can be reversed forcing it out from EI complex.
  • Since effective concentration of enzyme is reduced, the reaction velocity is decreased.
  • In competitive inhibition, the inhibitor will be a structural analog of the substrate.
  • There will be similarity in three-dimensional structure between substrate (S) and inhibitor (I). For example, the succinate dehydrogenase reaction is inhibited by malonate

In presense of competitive inhibitor, the MM equation becomes:

Clinical importance of competitive inhibition:

Enzyme Substrate Inhibitor Clinical Significance
Xantihine Oxidase Hypoxanthine Allopurinol Gout
Monoamine oxidase Catecholamines Ephedrine Amphetamine For elevated catecholamine
levels
Dihydrofolate reductase Dihydrofolic acid Aminopterin Methotrexate Treatment of leukaemia
and cancers
Acetylcholinesterase Acetylcholine Atropine,Non-depolarizing
muscle relaxants
In surgery for muscle relaxation
Tyrosine kinase tyrosine Imatinib and suntinib Anti cancer
Para Aminobenzoic acid (PABA)
Sulphanilamide Antibiotic
Vitamin K Dicumarol As anticoagulant
Pyridoxine Isonicotinic acid hydrazine Antituberculosis drug
NON-COMPETITIVE INHIBITION:
  • The inhibitor usually binds to a different domain on the enzyme, other than the substrate binding site.
  • Strong affinity for inhibitors prevent catalysis possibly due to distortion in enzyme conformation
  • A variety of poisons, such as iodoacetate, heavy metal ions (lead, mercury) and oxidizing agents act as irreversible non-competitive inhibitors.
  • Most probably the sites of attachment of the substrate and inhibitor are different.
  • The inhibitor binds reversibly with a site on enzyme other than the active site.
  • So the inhibitor may combine with both free enzyme and ES complex.
  • The velocity (Vmax) is reduced.
  • But Km value is not changed, because the remaining enzyme molecules have the same affinity for the substrate.
  • However, the kinetic properties in case of both are the same.
  • If the inhibitor can be removed from its site of binding without affecting the activity of the enzyme, it is called as Reversible-Non-competitive Inhibition.
  • If the inhibitor can be removed only at the loss of enzymatic activity by forming a covalent bond, it is known as Irreversible Non-competitive Inhibition.

Clinical importance of non-competitive inhibition:
  • Cyanide inhibits cytochrome oxidase.
  • Fluoride will remove magnesium and manganese ions and so will inhibit the enzyme, enolase, an consequently the glycolysis.
  • Iodoacetate would inhibit enzymes (Glyceraldehyde-3-P dehydrogenase, papain) having –SH group in their active centers.
  • Non competitive inhibitor of carbonic anhydrase is Acetazolamide
  • BAL (British Anti-Lewisite; Dimercaprol) is used as an antidote for heavy metal poisoning.
  • Disopropyl fluorophosphates (DFP) : Acetylcholinesterase enzyme cleaves acetylcholine to form acetate and choline and therefore terminates the action of acetylcholine. Disulfiram (Antabuse): Used in treatment of alcoholism, the drug irreversibly inhibits the enzyme aldehyde dehydrogenase
UNCOMPETITIVE INHIBITION:
  • Here inhibitor does not have any affinity for free enzyme.
  • Inhibitor binds to enzyme–substrate complex; but not to the free enzyme.
  • In such cases both Vmax and Km are decreased
  • Inhibition of placental alkaline phosphatase (Regan iso-enzyme) by phenylalanine is an example of uncompetitive inhibition.

Exam Question
  • The presence of a noncompetitive inhibitor can Leads to a decrease in the observed Vmax
  • A competitive inhibitor of an enzyme will Bind to the same site as the substrate
  • In competitive inhibition Vmax is unchanged but Km increased
  • Disopropyl phosphofluoridate (DFP) reacts with serine proteases irreversibly and therefore is Non competitive inhibitor
  • In noncompetitive antagonism Km value decreased; V max decreased
  • Atropine is useful in organophosphate poisoning because it Is a competitive antagonist of acetylcholine
  • Non competitive inhibitor of carbonic anhydrase is Acetazolamide
  • Non-depolarizing muscle relaxants are Competitive inhibitor of acetylcholine
  • Imatinib and suntinib are competitive inhibitors of tyrosine kinase
  • If V max decrease to 80% due to an inhibitor and Km is same as before Non competitive type of inhibition
  • Cyanide affects respiratory chain by Non-competitive irreversible inhibition
  • Substance which binds to substrate other than catalytic enzyme is Non-competitive inhibitor
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