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enzyme inhibitors-non covalent enzyme inhibitors.pptx
- 1. Enzyme inhibitors Dr.Shaheen Begum AssociateProfessor Sri Padmavati Mahila university, Tirupati
- 2. What we needto understand enzyme inhibition concept • Enzyme kinetics -It is the study of how enzymes bind substrates and convert them into products. It helps in understanding reaction rates, enzyme efficiency, and the effects of inhibitors. • Types of enzyme inhibitors: non-covalent and covalent inhibitors
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- 4. Details of theenzyme kinetics • K1=Rate constant for substrate binding; A higher k1means faster substrate binding • K2=Rate constant for substrate dissociation (breaking of the ES complex) • kcat(also called the turnover number) is a key kinetic parameter in enzyme reactions. It represents the number of substrate molecules converted into product by a single enzyme molecule per unit time when the enzyme is fully saturated with substrate. • A higher kcatmeans the enzyme can process more substrate molecules per second.
- 5. • Michaelis-Menten equationhelps in understanding enzyme efficiency and how enzymes behave under different substrate concentrations. • It relates substrate concentration with rate of the enzymatic reaction • The Michaelis-Menten equation is a hyperbolic curve, whereas the Lineweaver-Burk plot transforms it into a straight line for easier determination of KM and Vmax . • A low KMmeans the enzyme has a high affinity for the substrate, while a high KM means low affinity.
- 6. Classification of enzymeinhibitors
- 7. Inhibitor Type Binding Speed Reversibility BindingStrength Mechanism of Action Example Rapid Reversible Inhibitors Fast Fully reversible Weak to moderate (Kiin micromolar range) Binds quickly and dissociates easily; includes competitive, non-competitive, and uncompetitive inhibitors Ibuprofen (competitive inhibitor of COX enzymes) Tight Binding Inhibitors Fast Reversible but with very low Ki Very strong (Kiin nanomolar range) Binds enzyme tightly, often competing with substrate, but still reversible Methotrexate (inhibits dihydrofolate reductase) Slow Binding Inhibitors Slow Reversible Moderate to strong Takes time to form a stable enzyme-inhibitor complex, but can still dissociate FK506 (binds FKBP to inhibit immune response) Slow-Tight Binding Inhibitors Slow Initially reversible, but can become quasi-irreversible Extremely strong Forms an initial weak complex, then undergoes conformational changes leading to tighter binding Ritonavir (HIV protease inhibitor) Multisubstrate Analogs Varies Reversible Moderate to strong Mimics multiple substrates, blocking multi-substrate enzyme function PALA (mimics ATP and carbamoyl phosphate in aspartate transcarbamoylase) Transition State Analogs Fast Irreversible or extremely tight Very strong (Kiin picomolar range) Mimics the high-energy transition state of the substrate, stabilizing enzyme binding and preventing catalysis Penicillin (mimics transition state in bacterial transpeptidase)
- 8. Rapid reversible inhibitors •This class of inhibitors acts by binding to the target enzyme's active site in a rapid, reversible, and noncovalent fashion. • The net result is that the active site is blocked and the substrate is prevented from binding. • There are 3 types • Competitive • Uncompetitive • Non competitive
- 9. Types of rapidreversible inhibition
- 10. the kinetic signatureof competitive inhibitors: with increasing inhibitor concentrations, KM is increased but Vmax is unaffected. Non-competitive inhibitors: Vmax is reduced and the Km is not affected. Uncompetitive inhibitors: Vmax and Km are decreased
- 12. Competitive inhibition This reactionis competitively inhibited by malonate (-00CCH2C00-) that has, like succinate, two carboxylate groups. It is therefore able to bind to the enzyme's active site but, with only one carbon atom between the carboxylates, further reaction is impossible. Substrate analogs are rarely useful as enzyme inhibitors, given that large concentrations are required for inhibition, and their inhibition is readily overcome by any buildup of substrate. However, they are often useful probes for determining enzyme specificity and even mechanism.
- 13. Uncompetitive inhibition/ anti-competitive inhibition •takes place when an enzyme inhibitor binds only to the complex formed between the enzyme and the substrate (the E-S complex). Uncompetitive inhibition typically occurs in reactions with two or more substrates or products
- 14. Example: cyanide noncompetitivelyinhibits cytochrome c oxidase, the last enzyme in the electron transport chain. It leads to cyanide toxicity Heavy metals such as mercury, lead bind with enzymes in a non- competitive manner
- 15. Noncompetitive inhibition It isa type of allosteric regulation, is a specific type of enzyme inhibition characterized by an inhibitor binding to an allosteric site resulting in decreased efficacy of the enzyme. • An allosteric site is simply a site that differs from the active site- where the substrate binds. • In this inhibition, the inhibitor binds at the allosteric site independently of substrate binding • the inhibitor does not compete with the substrate for active site binding. • Noncompetitive inhibition reduces the maximal rate of an enzyme’s catalyzed reaction while leaving the affinity of the enzyme for its substrate unchanged
- 17. Slow-binding inhibitors • Slow-bindinginhibitors are compounds that inhibit their target enzymes in a time- dependent manner. • Slow-binding inhibition is a phenomenon in which equilibrium between enzyme, inhibitor, and enzyme-inhibitor (EI)1. complex is established slowly on a time scale of seconds to minutes
- 18. Both slow-binding andslow-tight binding inhibitors interact with enzymes gradually, but they differ in binding strength, mechanism, and reversibility.
- 19. Arginase can beinhibited by slow binding inhibitors Arginase is a key metalloenzyme that catalyzes the hydrolysis of L-arginine into L-ornithine and urea. It plays a crucial role in several physiological processes
- 20. • Urea CycleRegulation: o Arginase is essential for nitrogen metabolism, especially in the liver, where it helps detoxify ammonia by converting arginine into urea for excretion. • Polyamine Synthesis: o The product L-ornithine is a precursor for polyamines (putrescine, spermidine, and spermine), which are essential for cell proliferation, differentiation, and tissue repair. • Nitric Oxide (NO) Regulation: o Arginase competes with nitric oxide synthase (NOS) for L-arginine, reducing NO production. NO is a critical molecule for vasodilation, immune response, and neurotransmission. • Cancer Progression & Immune Suppression: o Many cancers overexpress arginase to suppress immune responses by depleting L-arginine, which is essential for T-cell activation. o Arginase inhibitors are being explored as anticancer agents. • Neurodegenerative & Inflammatory Disorders: o Arginase overactivity is implicated in diseases like Alzheimer’s, asthma, pulmonary fibrosis, and arthritis.
- 21. Nitric oxide synthasealso acts on Arginine Arginase and nitric oxide synthase (NOS) are functionally interconnected because they both use L-arginine as a substrate. The balance between these two enzymes determines whether L-arginine is converted into urea and ornithine (via arginase) or into nitric oxide (NO) and citrulline (via NOS). This interplay has profound effects on vascular function, immune response, and disease progression. · Arginase and NOS compete for the same substrate: L-arginine. · If arginase activity is high, less L-arginine is available for NOS, reducing NO production. · If NOS activity dominates, more NO is produced, leading to vasodilation and immune activation.
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- 23. • Slow-binding inhibitorsinteract with the enzyme gradually, leading to a time-dependent inhibition that stabilizes an inactive enzyme-inhibitor complex. These inhibitors are important for sustained therapeutic effects • Hydroxyarginine Derivatives o Compounds like N-hydroxy-L-arginine (NOHA) act as transition-state analogs, binding slowly but strongly to the enzyme. o They are promising for cardiovascular and immune- modulatory therapies.
- 24. Tight-binding inhibitors • Tight-binding,reversible inhibitors have dissociation constants between irreversible inhibitors and the highly reversible ones. Tight-binding inhibitors are those of which a significant fraction of the total inhibitor binds to the enzyme in order to observe intermediate levels of inhibition.
- 25. Inhibitors classified onthe basis of structure /mechanism Reactants and products are called as ground states of the enzymatic reaction Compounds that mimic the substrates and block the enzymatic reaction are substrate analogs Ground state analogs, multisubstrate analogs, translational analogs