Enzyme Inhibition and Regulation of Enzyme Activity

Enzyme Inhibition and Regulation

Enzymes . Inhibition of enzymatic activity . Drugs that act as inhibitors of enzyme activityEnzymatic inhibition In the kinetic discussion of enzyme inhibition the assumptions of Michael-Menten also apply to the enzyme KI E+I EI Any substance that reduces the velocity of an enzymatic reaction is to be considered an INHIBITOR. The inhibition can be IRREVERSIBLE or REVERSIBLE (competitive, non-competitive, uncompetitive)It is IRREVERSIBLE when the inhibitor and the enzyme bind very tightly to form the EI complex and the back reaction is very slow or does not occur at all, because the enzyme is inactivated and is no longer able to bind the normal substrate. Or when the irreversible inhibitor dissociates very slowly from its target enzyme because it is tightly bound to the enzyme, either covalently or non-covalently A REVERSIBLE inhibitor forms non-covalent and transient bonds with the enzyme. The reversible formation of a non-covalent bond with a molecule other than the substrate leads to the formation of anomalous, non-productive complexes which are not part of the normal catalytic process.

Irreversible Enzyme Inhibition

• NERVE AGENTS (nerve gases) are a group of synthetic substances whose production began in the 1930s by some German industries looking for new insect repellent.
• poisonous gas used in war as a weapon They are ACETYLCHOLINESTERASE enzyme inhibitors
• They bind to the enzyme with stable (covalent) bonds . They cannot be removed by simple means . They can be used - in the study of catalytic sites - as drugs (anti-metabolites) - as pesticide

The enzyme acetylcholinesterase (AchE) is responsible for the cleavage of acetylcholine (Ach) into choline and acetic acid acetylcholine + H2O -> choline + acetate ACETYLCHOLINE is the neurotransmitter at neuromuscular junctions, at synapses in the ganglia of the visceral motor system, and at a variety of sites within the central nervous system

Acetylcholine Receptors and Breakdown

Acetylcholine Receptors Transmitting Neuron Receiving Neuron O O Acetylcholinesterase Fig. 1. After signalling, acetylcholine is released from receptors and broken down by acetyl- cholinesterase to be recycled in a continuous process.HO-C- CH3 Acetylcholine Acetic Acid CH CH 3 CH3 + N-CH,CHO-C-CH3 + Electrostatic attraction CH CH, 13 3CH3 HO Choline Si I N 1 Serine "Esteric Site" CH2 CH2OH Acetylcholinesterase . The active site of the acetylcholinesterase enzyme is located in a very narrow pocket in which there are three amino acids serine, histidine, glutamate. . The amino acid serine is considered the target of attack by these NERVE AGENTS which irreversibly bind to it and thus block the enzyme, preventing it from functioning. e Anionic

Nerve Agents: Molecular Mechanisms

NERVE AGENTS (molecular mechanisms) The compound diisopropyl fluorophosphate (DFP), irreversibly inhibits biological systems by forming an enzyme-inhibitor complex with a specific OH group of the serine located in the active sites of these enzymes. 0 + F-P-OCH(CH3)2 I OCH(CH3)2 CHO-OCHCh + HF OCH(CH3)2 0 Nerve agents, such as DFP, inhibit the active site of the acetylcholinesterase enzyme

Other Examples of Irreversible Inhibition

Other examples of irreversible inhibition Organophosphorus (OPs) are among the most widely used insecticides in agriculture, in veterinary medicine, but also in domestic and work environments, thus causing great hazards to human health OPs constitute an extremely vast class of compounds with different chemical-physical properties, all sharing the same mechanism of action: the irreversible inhibition of the enzyme acetylcholinesterase.

Reversible Inhibition of Enzymatic Activity

REVERSIBLE INHIBITION of enzymatic activity A) Competitive inhibition A competitive inhibitor is a substance that binds to the free enzyme, thus preventing the formation of the enzyme-substrate complex (ES). It may be an analogue of the substrate, an alternative substrate for the enzyme or a reaction product that are not transformed B) Non-competitive (or mixed) inhibition In a typical non-competitive inhibition system, the inhibitor has no effect on the binding of the substrate to the enzyme, and the substrate has no effect on the formation of the inhibitor- enzyme bond, as both bind reversibly to the enzyme, at different sites C) Uncompetitive inhibition The typical uncompetitive inhibitor is a substance that binds to the ES complex giving rise to the non-productive ESI complex. These three types of inhibition differ in THE NATURE OF THE INTERACTION between the enzyme and the inhibitor and in the INHIBITOR'S EFFECT on enzyme kinetics

Competitive Inhibition Details

Competitive A competitive inhibitor can only bind to free enzyme (E). In the classical model, substrate and inhibitor compete for the same site on the enzyme. The presence of the inhibitor has an effect only on the Km, which increases as [I] increases: the affinity of the enzyme for the substrate decreases. The Vmax instead remains unchanged: in fact, for any [I] there is a [S] able to "cancel" the inhibitory effect. This is evident, considering that an increase in [S] shifts the equilibrium of the system towards the formation of ES.E + S ES - E + P + I S S - EI 1 I I Competitive inhibition

Competitive Inhibition Graphs

Representation of COMPETITIVE INHIBITION according to the graphs of MICHAELIS-MENTEN and LINWEAVER-BURK Vo 1/Vo 4 A Vmax 1 inhibitor Increase Km and inhibitor Vmax remains unchanged Vmax/2 1/Vmax ->KM [S] ->1/KM 1/[S]

Competitive Inhibition: Methanol Poisoning Treatment

Competitive inhibition: the ethanol treatment of methanol poisoning >In methanol poisoning, methanol is acted upon by alcohol dehydrogenase. >It is converted to formaldehyde which leads to toxicity. >This leads to metabolic acidosis, retinal oedema, COMA and death. > For treatment ethanol is used as it can competitively bind to alcohol dehydrogenase. · Medical application Methanol Formaldehyde Tissue damage e.g. blindness Alcohol dehydrogenase in liver Ethanol Acetaldehyde CH3CH2OH+ NAD+ -> CH3CHO + NADH + H+ . Mitochondrial: low Km Acetaldehyde dehydrogenase · Cytosolic: high Km CH3COO- + NADH + 2H+ Ethanol can be used as a competitive alcohol dehydrogenase inhibitor in cases of methanol poisoning. If used promptly it removes methanol from the active site of the enzyme

Competitive Inhibition: Succinic Dehydrogenase Example

The competitive inhibitor is a substance that binds to the free enzyme, thus preventing the formation of the ES complex. It may be an unmetabolizable analogue of the substrate, an alternative substrate for the enzyme or a reaction product. The inhibition of succinic dehydrogenase by malonic acid is a classic example of competitive inhibition by an unmetabolizable analogue: 0 Example of COMPETITIVE INHIBITION Substrate analogue inhibition 0 C O 0 0 succinic acid malonic acid succinic dehydrogenase O 0 0 0 fumaric acid

Competitive Inhibition Characteristics

Competitive Inhibition O CH 11 HỒ 0- CH Fumarate CH. T CH, CH, - O C 0=9 o Enzyme Succinate Succinate-enzyme complex (a) 1 11 ÇH2 1 C. - C 0 Enzyme Malonate Malonate-enzyme complex (b) The Vmax is not altered, since if the substrate concentration is very high, this is able to completely displace the inhibitor from the active site and to react with the same speed as in the absence of the inhibitor This fact decreases the affinity of the enzyme for the substrate: consequently the Km of the enzyme increases since a higher concentration of substrate is required to obtain half of the Vmax No product is formed O O-C-CH2 C + Enzyme 0=c

Uncompetitive Inhibition Details

Uncompetitive inhibition An uncompetitive inhibitor can only bind to the enzyme-substrate (ES) complex. The binding of the substrate to the free enzyme induces a conformational change which makes the site accessible for the inhibitor. An uncompetitive inhibitor affects both Vmax (which decreases) and Km. In contrast to competitive inhibition, Km decreases with increasing [I]: apparently the affinity of the enzyme for the substrate increases. Uncompetitive inhibition is therefore more marked at high concentrations of substrate: increasing [S], in fact, increases [ES], the form to which the inhibitor binds. An example: glyphosate herbicide (Roundup®) is an uncompetitive inhibitor of aromatic amino acids biosynthesis pathway. The plant dies because it lacks these amino acids.E + S ES > E+ P + I S S K1 ESI I I S

Uncompetitive Inhibition Graphs

Representation of UNCOMPETITIVE INHIBITION according to the graphs of MICHAELIS-MENTEN and LINWEAVER-BURK The inhibitor binds only to the enzyme-substrate complex, therefore Km and Vmax decrease 1/v enzyme + inhibitor M/Vmax 2 enzyme without inhibitor 1/[ $ ]

Non-Competitive (Mixed) Inhibition

Non-competitive (or mixed) inhibition In a typical non-competitive inhibition system, the inhibitor has no effect on the binding of the substrate to the enzyme, and the substrate has no effect on the formation of the inhibitor- enzyme bond, as both bind reversibly to the enzyme, at different sites Some non-clinically relevant toxic compounds are non-competitive inhibitors, given the frequency of the relative intoxications. Among these the heavy metals LEAD and MERCURY which inhibit the numerous enzymes whose activity is dependent on the integrity of the SH groups va + B + St substrate enzyme non-competitive inhibitor catalyzed reaction non-competitive inhibition ES complex - EI complex + A + B reaction products + unreacted substrate enzyme + 4 A

Non-Competitive Inhibition Graphs

Representation of NON-COMPETITIVE INHIBITION according to the graphs of MICHAELIS-MENTEN and LINWEAVER-BURK Vo 1/Vo A 4 inhibitor 1 inhibitor ĮV max Vmax decreases and Km remains unchanged max KM [S] 1/KM 1/[S] LV max/2

Enzyme Inhibitors as Drugs

Numerous molecules that act as ENZYME INHIBITORS are used as DRUGS Examples of drugs that act as enzyme inhibitors antibiotics; anti-inflammatoi treatment of hypercholesterolemia; a-glycosidase enzyme inhibitors (antidiabetics) treatment for uric acid increase

How Drugs Inhibit Enzymes

How do drugs work in enzyme inhibition? Active Enzyme Substrate Product Cellular Function Some drugs INTERACT with enzymes by blocking their enzyme activity Inactive Enzyme Substrate - Bound Enzyme Inhibitor (Drug) 100001

Irreversible Inhibition of Bacterial Transpeptidase by Penicillin

Irreversible inhibition of the bacterial enzyme transpeptidase Role of PENICILLIN Penicillin irreversibly inactivates glycopeptide transpeptidase, a key enzyme in bacterial wall peptidoglycan synthesis. The conformation of the penicillin can easily enter the active site of the enzyme where it binds very stably, this binds covalently and therefore irreversibly to the active site of the enzyme itself, inhibiting bacterial wall synthesis. (A) variable group 0 C H HN. S .CH3 C N CH3 0 coo" reactive peptide bond of the lactam ring penicillin R1 IZ N H 1 S CH3 5 O 2 IN 3 O 4 CO2H + : OH I Ser I penicillin bound to transpeptidase R1 IZ S CH3 O CH3 O CO2H :0: I Ser I enzyme enzyme CH3 IZI N =