Enzyme Inhibition: A Comprehensive Study

Enzymes Enzymes are specific molecules, act on one or few types of molecules (substrates) to give some type of molecular product. Biological catalysts: capable of accelerating a chemical reaction without being part of the final products or being consumed in the process. This binding of the...

Enzymes Enzymes are specific molecules, act on one or few types of molecules (substrates) to give some type of molecular product. Biological catalysts: capable of accelerating a chemical reaction without being part of the final products or being consumed in the process. This binding of the substrate forces the enzyme to change conformation (oxygen binding to Hb) This decreases the energy of activation needed to change the substrate to a transition state (unstable) and that allows the reaction to proceed to form a product and regenerate the enzyme  Regulation of Enzyme Activity 1. Covalent modification: A process in which a chemical group is covalently added to removed from the protein (phosphorylation) 2. Allosteric regulation/Feed back inhibition: end product of metabolic reaction produced in excess inhibits the first or regulatory enzyme in the sequence 3. proenzymes: the inactive form of enzyme which can be activated by removing a small part on their polypeptide chain  Factors that affect the activity of enzyme ▪ pH, ▪ temperature, ▪ substrate concentration, ▪ enzyme concentration, ▪ presence of inhibitors or activators  Michaelis-Menten hypothesis Model proposed by Michaelis and Menten: the enzyme reversibly combines with its substrate to form ES complex that subsequently yields product, regenerating the free enzyme k kcat E+S f ES E+P kr Variables [E]: free enzyme molecules Parameters [S]: free substrate molecules kf , kr , kcat : reaction rates [ES]: enzyme-substrate complexes [P]: free product molecules  Michaelis-Menten Equation It describes how reaction velocity varies with [S]. efficiacy Potency potency Km (Michelis constant): is defined as the [S] at which the reaction rate is half Vmax (1/2 Vmax). It is characteristic of an enzyme and reflects its affinity to the substrate: high Km – low affinity Low Km – high affinity Velocity of the reaction: is proportional to enzyme concentration (because[S] is not limiting  Lineweaver-Burk Plot straight line When it is difficult to determine Vmax and hence the Km of a reaction from the plot, you should plot the reciprocal of [S] against the reciprocal of the velocity (1/v against 1/[S]) This changes the hyperbolic curve into a straight line, which intercepts the x-axis at - 1/Km and the y-axis at 1/Vmax. This plot is known as: Lineweaver-Burke plot (double reciprocal plot): 1 Km 1 1 = + v Vmax [S] Vmax  Types of enzymes inhibitors Enzyme inhibitors: molecules & factors that interact with the enzyme, and prevent (block) it from working. Classified into: - Nonspecific Inhibitors: These effect all enzymes in the same way (e.g. acid, alcohol…) - Specific Inhibitors: These exert their effects on a single enzyme. They are classified into: Reversible: the inhibitor can dissociate from the enzyme, and catalytic activity is regained: Competitive: resemble the enzyme’s substrate and compete with the substrate molecules for the active site. Non-competitive: do not resemble the enzyme's substrate, thus do not bind enzyme at the active site but at other site and inducing enzyme to undergo conformational changes. Uncompetitive: bind only with the ES form of the enzyme Irreversible: the inhibitor combine with the functional groups of the amino acids in the active site, irreversibly.  Competitive inhibitors Reversible ▪ Inhibitor is structurally similar to the substrate ▪ Inhibitor binds reversibly to the same site that the substrate binds competes with the substrate binding ▪ Inhibitor can be reversed by increasing the concentration of substrate reversible  Competitive Enzyme Inhibition The Km increases: affinity of the enzyme towards substrate is decreased in presence of the inhibitor Vmax remains unchanged: Increasing [S] will displace the inhibitor and allows the reaction to reach Vmax umax not affected affinity increased  STATIN Competitive inhibitors: Clinically useful competitive inhibition (STATINs)  Non-competitive Inhibitors ▪Inhibitor binds at a site other than the active site of the enzyme ▪Inhibitor has no structural resemblance to the Substrate-No competition for binding ▪Increase of substrate concentration does not relieve this inhibitor stopped ▪Reaction is slowed down but not halted  Non-competitive Inhibition Km value is unchanged inhibitor do not interfere with the binding of substrate to the enzyme Vmax decreases inhibitor can not be overcome by increasing the concentration of substrate  Non competitive inhibitors: Clinically useful competitive inhibition BAL (British Anti-lewisite; Dimercaprol) is a medication used as an antidote for poisoning by heavy metals (mercury, arsenic, lead…): Heavy metals acts as enzyme poisons by reacting with SH group of the enzyme; BAL has several SH groups with which the heavy metals ions can react and thereby their poisonous effects are reduced.  Uncompetitive binds Inhibitors to complex not , enzyme ▪Inhibitor binds only to the ES complex, not to free enzyme Enzyme become catalytically inactive ▪Inhibitor can’t be reversed by increasing the concentration of substrate since the inhibitor doesn’t compete with the substrate for the same binding site Decrease in both Vmax and Km +I Example: placental alkaline phosphatase (ALP) inhibited by phenylalanine +I  Irreversible Inhibition Inhibitors bind to enzymes (functional groups in the active site of the enzyme) very tightly through covalent or non-covalent bonds (irreversible) enzyme Inhibition Slow dissociation of EI complex that’s why it is considered irreversible. A variety of poisons such as organophosphates (OP) poisoning and oxidizing agents act as irreversible inhibitors. Irrevirsible inhibitor chibitor of organophosphate  Irreversible Inhibition ▪In term of kinetics, irreversible inhibition is similar to non competitive inhibition: Vmax decreased Km unchanged  Cholinesterases breaks dow ach to acetyl COA (ChEs) and choline Enzymes needed for the proper functioning of the nervous systems of humans, other vertebrates and insects. 1. How does it work? Stimulating signals are usually carried by acetylcholine (neurotransmitter). These signals are discontinued by a specific cholinesterase called acetylcholinesterase which hydrolyze the acetylcholine into choline and acetic acid. These reactions are necessary to prevent acetylcholine reactivating receptor and allow the neuron to return to its resting state after activation. active inactive Hydrolysis reaction 2. Types of cholinesterase The main type is: acetylcholinesterase (AChE) also called choline esterase I or erythrocyte cholinesterase or RBC cholinesterase or acetylcholine hydrolase It is found mainly in neural synapses, neuromuscular junctions, CNS and RBC membranes. The other type is: butyrylcholinesterase (BuChE) also called choline esterase II or pseudocholinesterase or plasma cholinesterase; it is produced in the liver ( BuChE levels may be reduced in patients with advanced liver disease) and found mainly in the blood plasma. Both AChE and BuChE are inhibited by some organophosphate (OP) and carbamate (CB) esters and also by other chemicals.  Anti-cholinesterases (cholinesterase inhibitors) Agents which inhibit cholinesterase enzyme / Block hydrolysis of acetylcholine causes able to reactivate cholinergic receptor and any Acetylcholine is · nerve impulses · are continually transmitted and muscle contraction do not stop (Same effect as a cholinergic agonist). 2 types of acetylcholinesterase inhibitors: Reversible: do not covalently modify acetylcholinesterase (combine with a residue of serine in the active site). Produce effects of moderate duration : E.g, carbamates, acridine Irreversible: organophosphates (phosphorylates the serine residue in the active site); produce effects of long duration (pesticide, sarin nerve gas)