Enzyme2 File

Enzymes Theories of enzyme substrate interaction 2 Theories of enzyme substrate interaction . There are two proposed methods by which enzymes bind to their substrate molecules: 1- Lock & key model 2-Induced fit model 3 Theories of enzyme substrate interaction 1-lock & key model (Template hypothesis for enzyme action): The substrate fits to binding site of enzyme as a key fits into proper lock . Enzyme & substrate possess specific complementary geometric & rigid shapes fit exactly into one another. This explains enzyme specificity, but fails to explain the stabilization of the transition state & allosteric regulation & inhibition. 4 1-lock & key model : 5 Theories of enzyme substrate interaction 2-Induce fit model More flexible model than lock & key model. Enzymes are flexible structures The active site modified as the [S] interacts with the anzyme (active site of enzyme become complementary to S) Model explains enzyme specificty & stabilization of transition state, regulation & inhibition. 6 2-Induce fit model 7 Enzyme Inhibition 8 Enzyme inhibitors: Any substance that can diminish the velocity of an enzyme-catalyzed reaction. 9 Types of inhibition Irreversible Reversible inhibitors inhibitors 10 Types of inhibition 1-Irreversible inhibitors : Bind to enzymes through covalent bond (inhibited enzyme does not regain activity upon dilution of enzyme-inhibitor complex) 11 Type of inhibition 2-Reversible inhibitors: Bind to enzymes through noncovalent bonds, thus dilution of the enzyme–inhibitor complex results in dissociation of the reversibly bound inhibitor, and recovery of enzyme activity. 12 Reversible inhibitors 1. Competitive inhibition 2. Non competitive inhibition 3. Uncompeptitive inhibition 13 1-Competitive inhibition The inhibitor and substrate compete for the same active site on the enzyme as a result of similarity in structure. The enzyme substrate complex will be broken down to products (E+S ES E+P) where as enzyme inhibitor complex;(EI) will not be broken down to product. products. 14 1-Competitive inhibition 15 16 Kinetics of Competitive inhibition Effect on Vmax: . The effect of a competitive inhibitor is reversed by increasing [S]. At a sufficiently high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of inhibitor (Vmax not changed) 17 Kinetics of Competitive inhibition Effect on Km: . A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve ½Vmax. 18 Kinetics of Competitive inhibition Effect on the Lineweaver-Burk plot: . Shows a characteristic Lineweaver-Burk plot in which the plots of the inhibited and uninhibited reactions intersect on the y-axis at 1/Vmax (Vmax is unchanged). The inhibited and uninhibited reactions show different x-axis intercepts, indicating that the apparent Km is increased in the presence of the competitive. 19 Kinetics of Competitive inhibition 20 Examples of competitive inhibitors Malonate that competes with succinate and inhibits the action of succinate dehydrogenase to produce fumarate in the Krebs cycle. 21 Examples of competitive inhibitors Antihyperlipidemic agents competitively inhibits the first committed step in cholesterol synthesis. This reaction is catalyzed by HMG-CoA reductase. • Statin drugs, such as atorvastatin (Lipitor) and simvastatin (Zocor), are structural analogs of the natural substrate for this enzyme, and compete effectively to inhibit HMG-CoA reductase. • By doing so, they inhibit de novo cholesterol synthesis, thereby lowering plasma cholesterol levels. 22 Statins 23 Examples of competitive inhibitors Allopurinol (used in treatment of gout) inhibits Xanthine oxidase by competing with Uric acid precursors for the active site on the enzyme. • This competition blocks the conversion of these precursors and of hypoxanthine and xanthine, to uric acid and result in lower serum urate levels. 24 25 Examples of competitive inhibitors The antibiotic sulfanilamide is similar in structure to para-aminobenzoic acid (PABA), an intermediate in the biosynthetic pathway for folic acid. • Sulfanilamide can competitively inhibit the enzyme that has PABA as it's normal substrate by competitively occupying the active site of the enzyme. 26 27 2-Noncompetitive inhibition • Noncompetitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme. • The noncompetitive inhibitor can bind either free enzyme or the ES complex, thereby preventing the reaction from occurring. 28 Noncompetitive inhibition 29 30 Kinetics of Noncompetitive inhibition Effect on Vmax: . Noncompetitive inhibition cannot be overcome by increasing the concentration of substrate. Thus, noncompetitive inhibitors decrease the apparent Vmax of the reaction. 31 Kinetics of Noncompetitive inhibition Effect on Km: . Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme. Thus, the enzyme shows the same Km in the presence or absence of the noncompetitive inhibitor. 32 Kinetics of Noncompetitive inhibition Effect on Lineweaver-Burk plot: . Noncompetitive inhibition is readily differentiated from competitive inhibition by plotting 1/vo versus 1/[S] and noting that the apparent Vmax decreases in the presence of a noncompetitive inhibitor, where as Km is unchanged. 33 Kinetics of Noncompetitive inhibition 34 Examples of noncompetitive inhibitors: Lead forms covalent bonds with the sulfhydryl side chains of cysteine in proteins. Ferrochelatase, an enzyme that catalyzes the insertion of Fe2+ into protoporphyrin (a precursor of heme), is an example of an enzyme sensitive to inhibition by lead. 35 Non competitve inhibitor may be reversible Irreversible: Reversible inhibitors Irreversible inhibitors Attach non covalent Covalent tightly bound Can dissociate. to the enzyme. 36 Irreversible inhibitors Irreversible inhibitor form permanent complexes with enzyme by make covalent bonds with enzyme. Inhibition cannot be reversed. 37 Irreversible inhibitors Nerve gas, inhibitors such as DIFP, (diisopropyl fluorophosphates) was very potent and toxic inhibitors of acetylcholinesterase, an enzyme involved in nerve action. If acetlycholine cannot be hydrolysed by this enzyme, nerve signals cannot be passed across the synapses of the nervous system. On exposure to this compound, death can result in minutes due to respiratory failure. 38 39 3-Uncompetitive Inhibition Uncompetitive Inhibitor binds only to ES complex at locations other than the catalytic Site, So formation of ESI alone. Substrate binding modifies enzyme structure, making inhibitor- binding site available. Inhibition cannot be reversed by substrate. In this case apparent Vmax and Km decreased. 40 3-Uncompetitive Inhibition 41 42 Kinetics of Uncompetitive Inhibition 43 Regulation of Enzyme Activity 44 Regulation of Enzyme Activity The regulation of the reaction velocity of enzymes is essential if an organism is to coordinate its numerous metabolic processes. Some enzymes with specialized regulatory functions respond to allosteric effectors or covalent modification, or they show altered rates of enzyme synthesis (or degradation) when physiologic conditions are changed. 45 A.Allosteric binding sites Allosteric enzymes: Enzymes have more than one site, where effector binding at one site induces a conformational change in enzyme altering its affinity for a substrate or modify the maximal catalytic activity of the enzyme. 46 A.Allosteric binding sites Allosteric enzymes are regulated by molecules called effectors (also called modifiers) that bind noncovalently at a site other than the active site. Effectors that inhibit enzyme activity are termed negative effectors, where as those that increase enzyme activity are called positive effectors. 47 Major features of allosteric enzymes 1) Multiple subunit . 2) Catalyze the committed step early in pathway. 3) Contain two seperate functional center: A)Catalytic B) Regulatory 4) May be subject to either +ve or –ve effectors that affect either Vmax or Km or both. 48 Major features of allosteric enzymes 5)Effectors may be coenzymes, substrate or product. 6) Undergo changes either in conformation or in the cooperativity of componant polypeptides by appropriate effector. 7) Don’t follow the kinetics of Michaelis Menten model. 49 A.Allosteric binding sites Effects of –ve or +ve effectors on an allosteric enzyme. A. Vmax is altered. B. The substrate concentration that gives half-maximal velocity (K0.5) is altered 50 A.Allosteric binding sites 1-Homotropic effectors: When the substrate itself serves as an effector. Most often, an allosteric substrate functions as a positive effector. In such a case, the presence of a substrate molecule at one site on the enzyme enhances the catalytic properties of the other substrate- binding sites that is, their binding sites exhibit cooperativity. • 51 A.Allosteric binding sites 2-Heterotropic effectors: The effector may be different from the substrate(Feed back inhibition). The enzyme that converts D to E has an allosteric site that binds the endproduct G. If the concentration of G increases (for example, because it is not used as rapidly as it is synthesized), the first irreversible step unique to the pathway is typically inhibited. 52 2-Heterotropic effectors: 53 B.Regulation of enzymes by covalent modification 1-Phosphorylation & dephosphorylation: . Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that use ATP as a phosphate donor. Phosphate

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