Enzymes SM modified 2024 Fall PDF

ENZYMES 1  What are enzymes? Enzymesare organic catalysts that increase the rates of chemical reactions without changes in the enzymes during the process; Enzymereactions occur under mild conditions, such as: Body temperature, Atmospheric pressure,...

ENZYMES 1  What are enzymes? Enzymesare organic catalysts that increase the rates of chemical reactions without changes in the enzymes during the process; Enzymereactions occur under mild conditions, such as: Body temperature, Atmospheric pressure, Neural pH , Most enzymes are highlysubstrate specific; Activity of an enzyme can regulated; Most enzymes are proteins, except a few catalytically active RNAmolecules(Ribozymes); 2 How is Activation Energyrelated to Transition state? For chemical reaction to proceed Energybarrier must be overcome (Fig.1), A+ B = AB C+ D Energy is needed to transform substrate into “Transitionstate (AB)”, Transition state has the highest “Free energy” of any component in the reaction pathway; “Gibbs” Free Energy of Activation (∆G‡) is the difference in Free energy between Transition state and Substrate (Fig. 1) 3 Fig.1: Diagramshowingenergychangesin Enzymereaction ∆G‡ = GibbsFree Energyof Activation; ∆G = Free Energyor GibbsFree Energy; 4 IsGibbsFree Energyof Activation affected by enzyme? Enzymedecreases Gibbs Free Energy of Activation; Enzymestabilizes Transition state of a chemical reaction; Enzymedoes not alter the energy levels of substrates and products; Enzymeincreases the rate at which reaction occurs, but hasno effect on overall change in energy of the reaction; Enzymeincreases the rate of chemical reaction by decreasing the Gibbs Free Energy of Activation (∆G‡) {SeeFig. 1}; 5 IsGibbsfree energyof activation (∆G‡) the sameasGibbsfree energy change( G)? Gibbs Free Energy change ( G) is different from Gibbs FreeEnergyof Activation (∆G‡) {Fig.1}; Gibbs Free Energy change ( G) is the free energy change between SUBSTRATE[S], ANDPRODUCT[P]; G= Free energyof [S] – Free energyof [P] Gindicates if a reaction is energetically favorable or not Gis independent of the path of the chemical reaction, Gprovides no information about the rate of a chemical reaction since the rate of the chemical reaction is governed by ∆G‡ 6  Negative Gindicates that the reaction is thermodynamically favorable in the direction indicated That it is likely to occur spontaneously, {Fig. 1}; Positive Gindicates that the reaction is not thermodynamically favorable and requires an input of energy to proceed in the direction indicated; Energy can be achieved by coupled reactions; Standard Free Energy change ( G°),defined [S] and [P] under specified biochemical conditions; 7 What isthe general equation for enzymecatalyzedreaction? General expression for an enzyme catalyzed reaction: E+ S+.=====èES======èE+ P (Where E= Enzyme;S=Substrate; ES= Enzyme-Substrate Complex;P= Product); Concept of “Active site” or “Catalytic site” or “Substrate-binding” site is needed to understand formation of ES-complex 8 What isthe Active site or Catalytic site of an enzyme? Active site or Catalytic site of an enzyme: Region that binds Substrate(s) and converts it into Product(s); Relatively small part of the whole enzyme molecule; Three-dimensional entity formed by amino acid residues that can lie far apart in the linear polypeptide chain; 9  Substrate binds in active site by multiple weak forces: Electrostatic interactions, Hydrogen bonds, Vander Waals bonds, Hydrophobic interactions, Reversible covalent bonds, Binding of Substrate to Active site gives the Enzyme-Substrate complex (ES); Catalytically active residues within the active site acts on Substrate, forming “Transition state” and then Products, which are released; 10 Briefly explain the Lock-and-KeyModel for Enzyme-Substrate binding Lock-and-KeyModel: (Fig. 2a) Proposed by Emil Fischer in 1894, According to this model the shape of the Substrate and the Active site on the enzyme fit together like a Keyinto its Lock; Both shapes are considered Rigid and Fixed, and perfectly complement each other when brought together in the right alignment; 11 Fig2a: Diagramof Lock-and-Keymodel Bindingof Substrate(S) to Enzyme(E) to form EScomplex 12 Briefly explain the Induced-Fit Model for Enzyme-Substrate binding Induced-Fit Model: (Fig. 2b) Proposed in 1958 by Daniel Koshland, Jr Binding of Substrate Induces a conformational changein the Active site of the enzyme; Enzymemay distort the Substrate, forcing it into a conformation similar to that of the “Transition state” 13 Fig2a: Diagramof Lock-and-Keymodel Bindingof Substrate(S) to Enzyme(E) to form ES-complex 14 Figure 6.4 - Two Models for the Binding of a Substrate to an Enzyme © 2018 Cengage Learning. Nomenclature of enzymes Someenzymeshave commonnames: Many enzymes are named by adding suffix “-ase” to the name of their substrate; Example: Urease:enzyme that catalyzes hydrolysis of Urea, Maltase: enzyme that catalyzes hydrolysis of Maltose, Someenzymes, such asTrypsinand Chymotrypsin, havenames that do not denote their substrate; Someother enzymes have several alternative names; 16 International Classificationof Enzymes International System of Nomenclature is to Standardized and Rationalize Names of Enzymes: Enzymesare place into one of SixMajor classesbased on the type of reaction catalyzed: Eachenzyme is identified uniquely by using a Four- Digit Classification number SixMajor classesare: Oxido-Reductases, Transferases Hydrolases Lyases, Isomerases, Ligasesor Synthases 17 Enzymes can be classified according to the chemical reactions they catalyze Oxidoreductases oxidation/reduction (eg. dehydrogenases) Transferases group transfer (eg. kinases) Hydrolases hydrolysis (eg. proteases) Lyases lysis, generating double bond (eg. synthases) Isomerases rearrangement (eg. racemases) Ligases ligation requiring ATP (eg. © 2018 Cengage Learning. synthetases) Enzymes fall into classes based on function There are 6 major classes of enzymes: Oxidoreductases: are involved in oxidation, reduction, and electron or proton transfer reactions Transferases: catalyze reactions in which groups are transferred Hydrolases: cleave various covalent bonds by hydrolysis Lyases: catalyze reactions forming double bonds Isomerases: catalyze isomerisation reactions Ligases: join substituents together covalently © 2018 Cengage Learning. © 2018 Cengage Learning. © 2018 Cengage Learning. © 2018 Cengage Learning. A zymogen, an inactive precursor of an enzyme, can be irreversibly transformed into an active enzyme by cleavage of covalent bonds. © 2018 Cengage Learning. Another class of proteases is the caspases, which are a family of homodimer cysteine proteases responsible for many processes in cell biology, including programmed cell death, or apoptosis. © 2018 Cengage Learning. Abzymes: antibodies that are produced against a transition-state analog and that have catalytic activity similar to that of a naturally occurring enzyme. © 2018 Cengage Learning. © 2018 Cengage Learning. © 2018 Cengage Learning. © 2018 Cengage Learning. © 2018 Cengage Learning. © 2018 Cengage Learning. © 2018 Cengage Learning. © 2018 Cengage Learning. What isthe velocity or rate of an enzymereaction? Velocity or Rate of Enzyme-catalyzed reaction is the changein the amount of Substrate or Product per unit time; Velocity of Enzymeis measured under “Steady- State” conditions, when the amount of Substrate is very large compared to amount of enzyme; Velocity is reported asthe value at time zero (Vo); Initial Velocity (Vo) and is expressed as µmol/min Velocity is fastest at time zero, which is the point when no product has been formed (Fig. 3) 34 Fig.3: Diagram showingAmount of Productformed and Time for an enzymecatalyzedreaction {Vo= Initial Velocity} 35  Fig.3 shows Typical graph of Product formed against Time for an Enzyme-catalyzed reaction; Initial period of rapid product formation gives the linear portion of the graph; Slowing-down of the enzyme velocity asSubstrate is used up and/or asthe enzyme loses activity; Initial Velocity (Vo) is obtained by drawing a straight line through the linear part of the curve, starting at the zero time-point Slope of the straight line is equal to Vo 36 How is the activity of an enzymeexpressed(Enzymeunits)? An enzyme unit (U) is the amount of enzyme that catalyzes the transformation of 1.0µmol of Substrate per minute at 25°Cunder optimal conditions for that enzyme; Activity of an Enzyme,is the Total Units of Enzymein the sample, Specific Activity is the Number of Units per milligram of Protein (units/mg) Specific Activity is a measure of the purity of an enzyme; During the purification of an enzyme, the Specific Activity increases and becomes maximal and constant when the enzyme is pure 37  How doesEnzymeconcentration affect reaction velocity? When Substrate concentration is constant, the Vo is directly proportional to the concentration of the Enzyme, Increasing the amount of Enzymeincreases Vo; Straight-line graph is obtained when Vo is plotted against Enzymeconcentration; 38  How does[S] affect velocity of enzyme reaction? When Vois plotted against [S] and Hyperbolic curve is obtained (Fig.3a) Thecurve can be separated into sections based on concentration of the Substrate [S] (Fig.3b) At low [S]: Doubling of [S] will lead to doubling of the Vo, It is a “First-order” reaction; At higher [S]: Enzymebecomes saturated, Increase in [S] lead to very small changes in Vo, It is a “Zero-order” reaction; 39 Fig3a: Graph of [S] vsVo For constant amount of Enzyme[E]; Graph [S] vs Vogives Hyperbolic curve; Curve has three sections, based on [S]; (see Fig. 3b) 40 Fig.3c: Graph of [S] against Vo; Diagram shows order of reactions: Firstorder, Mixed order, Zeroorder with respect to [S] 41  What isa First-order reaction? Firstorder reaction: For a given amount of enzyme, the Velocity of the reaction is directly proportional to the [S]; Increasing the [S] also causeincrease in Velocity of reaction; Relationship between Velocity and [S] is Linear 42  What isa zeroorder reaction? Zeroorder reaction: For a given amount of Enzyme[E], the Velocity of the reaction is Constant and Independent of Substrate [S] concentration; Increasing the Substrate concentration [S] has no effect on reaction velocity; Addition of more substrate will not speed up the reaction; 43 Fig3b: Graphshowingrelationship between [S] and Vo: Graphcanbe separated into 3 sections: At low [S]: reaction is First-order with respect to [S]; Vois directly proportional to [S] At mid [S]: reaction is Mixed-order; Proportionality of Voto [S] is changing; At high[S]: reaction is Zero-order, Vois independent of [S] Enzymehas its maximum velocity (Vmax); Increasing [S] has no effect on Vo; enzyme is saturated; These enzymes are called Michaelis-Menten enzymes 44 How doesTemperature (T) affect rate of enzyme reaction? T °Caffects rate of enzyme reactions in two ways: First: Risein T °Cincreases rate (Vo) of reaction; Second:Increasing T °Cabove a certain value causes inactivation of enzyme (denature of enzyme) and thus reducesthe rate of reaction; Most enzymes are Thermo-labile, In Hypothermia most enzyme reactions are depressed, causing reduction in metabolism; Graph of T °Cagainst Vogives a curve with well-defined optimum T°C (Fig. 4a) Temperature optimum: about 37°C, 45 Fig.4: Graph of T °Cagainst Vogives a curve with well- defined optimum T°C 46 How does pH affect rate of enzyme reaction? Eachenzyme has an Optimum pH at which rate of reaction is maximum; Smal deviations in pH from optimum value lead to reduced rate caused by changes in ionization of groups at the active site of enzyme; Larger deviations in pH lead to denature of enzyme due to interference with weak non-covalent bonds that maintaining the three-dimensional structure; Optimum pH of most enzymes is around 6.8, but there is diversity in pH optima, due to different functions; Digestive enzyme Pepsin has acidic pH of stomach (pH 2) 47 Fig.4b: Graphof VoagainstpH for two different enzymes; Eachgraph is bell-shapedcurve; 48  ENZYMEKINETICS General expressionfor an enzyme catalyzed reaction: E+ S+.=====èES======è E+ P (Where E= Enzyme;S=Substrate;ES= Enzyme-SubstrateComplex; P= Product) What doesMichaelis-Menten equation represent? Relationship between Initial velocity (Vo) and substrate concentration [S]; 49 What is the Michaelis-Menten equation? Michaelis-Menten equation: Vo= Initial velocity, Vmax= Maximum velocity, [S] = Substrate conc. Km= Michaelis constant, 50 What are the 3 basicassumptionsof Michaelis-Menten equation? 1. [S] is very large compared to [E]; Enzyme is saturated with substrate, no free enzyme is available; 2. [ES]complex is in a “Steady-State”, (i.e., Rate of formation of EScomplex is equal to its rate of breakdown); 3. Initial velocity (Vo) of reaction must be used; 51  What isKm? Kmis equal to the [S]at which the Initial velocity (Vo) is equal to half the Maximal velocity, That is: Km= [S], when Vo= 0.5 Vmax Kmis a measure of the stability of ES-complex 52  Enzyme Mechanisms Common mechanisms for enzymes that catalyze reactions containing two or more substrates are: Ordered mechanism Substrates have to bind to the enzyme in a specific order Random mechanism Substrates can bind to the enzyme in any order Ping-pong mechanism Substrate binds to the enzyme and releases a product before the second substrate binds to the enzyme © 2018 Cengage Learning. Ordered and Random Mechanisms - Examples Ordered mechanism Random mechanism © 2018 Cengage Learning. Ping-Pong Mechanism - Example © 2018 Cengage Learning.  How significant is Km? Smallvalue of Km,means highaffinity of the enzyme for the Substrate; Largevalue of Km,means low affinity of enzyme for the Substrate; Kmis characteristic for a particular enzyme with a given substrate; Kmis an important parameter in metabolic control in cells; Kmvalues are near to the concentration of substrates in cells; 56 What is Lineweaver-Burk Plot (Double-Reciprocal Plot)? Lineweaver-Burk Plot (Double-Reciprocal plot) is used to determine Kmand Vmaxfor an enzyme; Michaelis-Menten equation is rearranged to give Double-reciprocal equation; Equation is similar to that for a straight-line: Y= MX + C, 37  Equation is used to plot the Double-reciprocal graph Information that can be obtained from graph (Fig. 5): Y= MX + C Slopeof graph:M = Km/Vmax, Intercept on Y-axis:C= 1/Vmax, Intercept on X-axis:when 1/Vo = 0, gives:– 1/Km 58 Fig.5a: Graph of [S] against Vo,show how to obtain Km; Fig.5b: Lineweaver-Burk plot (double-reciprocal plot) showing how to obtain Km F 59  ENZYMEINHIBITION Somecompounds can inhibit enzyme reactions; Twomain types of Enzymeinhibition are: Reversible Inhibition, Irreversible Inhibition, Reversible inhibition can be subdivided into: Competitive inhibition, Non-competitive inhibition, 60  What is competitive inhibition? Competitive inhibition: Substrate [S] and Inhibitor [I] competes for the binding site of the enzyme; Competitive Inhibitor: Hasstructural similarities to the Substrate, Competes with substrate for enzyme active site, Binds reversibly to active site of Enzyme, Enzymemay bind either Substrate [S] or Inhibitor [I], but not both at the same time; High [S] displaces competitive inhibitor from active site; Fig.6 illustrates binding of [S] and Inhibitor [I] to binding site of Enzyme[E]; 61 Competitive vs Noncompetitive inhibition 62  Competitive inhibition Inhibitor binds only to unoccupied Enzyme Vmax remains unchanged, Km increases ([S] is more ‘diluted’, less [E]) 63 Fig.6a: Bindingof [S] and [I] to bindingsite of Enzyme[E] 64 Fig.6b: In competitive inhibition Enzyme(E) canbind to either Substrate[S] or Inhibitor [I]; it cannot bind to both at the sametime. 65  Give examples of Competitive Inhibition Sulfanilamide competes with Para-Aminobenzoic Acid (PABA)in reaction catalyzed by DihydropteroateSynthetasein the biosynthesis of Folate (Folic Acid); Methotrexate competes with Dihydrofolate in reaction catalyzed by Dihydrofolate Reductase (Methotrexate is structural analogue of Folate) 66 How canMichaelis-Menten Constant(Km) be determined in Competitive Inhibition of an enzyme? Measure Voat different [S] in the absence and presence of a fixed concentration of the Competitive Inhibitor [I]; Draw Lineweaver-Burk plot for Inhibited and Non- inhibited enzyme (Figs.7); Interpretation of the results: There is no changein Vmaxof the Enzyme, The Kmof the inhibited Enzymeincreases, Competitive inhibitor increasesthe Kmof an enzymefor its substrate; 67 Fig. 7: Diagram of Lineweaver – Burk (Double-Reciprocal) plot showing effect of Competitive Inhibitor on Km and Vmax of an Enzyme. 68  Competitive inhibition It is important to remember that the most distinguishing characteristic of a competitive inhibitor is that substrate or inhibitor can bind the enzyme, but not both. Because both are vying for the same location, sufficiently high substrate will “outcompete” the inhibitor. This is why Vmax does not change; it is a measure of the velocity at infinite [substrate]. 69 Competitive inhibitors mimic substrates Rational Drug Design substrate Purine nucleoside phosphorylase Potent inhibitor 70 What is Non-Competitive inhibition (Give example)? Non-competitive inhibitor binds reversibly to a site other than Active Site of the enzyme, (Fig.8a); Enzymecan bind Inhibitor, Substrate or both the Inhibitor and Substrate together (Fig.8b); Effects of non-competitive inhibitor cannot be overcome by increasing [S]; Example of Non-competitive inhibitor: Inhibition of Renin by Pepstatin 71 Noncompetitive (mixed) inhibition Inhibitor binds to both unoccupied Enzyme and Enzyme- Substrate (E-S) complex Vmax decreases, Km remains the same 72  Noncompetitive inhibition With a pure, noncompetitive inhibitor, the binding of substrate does not affect the binding of inhibitor, and vice versa. Because the K is a measure of the affinity of the M enzyme and substrate, and because the inhibitor does not affect the binding, the K M does not change with noncompetitive inhibition. 73 Uncompetitive inhibition Inhibitor binds only to Enzyme-Substrate (E-S) complex Vmax decreases, Km decreases(Le Chatteliar’s Principal) 74  Uncompetitive inhibition This reduction in the effective concentration of the E-S complex increases the enzyme's apparent affinity for the substrate through Le Chatelier's principle (Km is lowered) and decreases the maximum enzyme activity (Vmax), as it takes longer for the substrate or product to leave the active site. 75 Fig.8a: 76 Fig.8b: 77 How canMichaelis-Menten constant be determined for Non-Competitive Inhibition of an enzyme? Measure Vo of the enzyme at different [S] in the absence and presence of a fixed concentration of non-competitive inhibitor; Draw Lineweaver-Burk plot for Inhibited and Non-inhibited enzyme (Fig.9) Interpretation of results: Km is not affected, thus affinity of the enzyme for Substrate in unchanged; Vmaxof inhibited enzyme is decreased; 78 Fig.9: 79  What is Irreversible inhibition (Give examples)? Irreversible Competitive inhibitors inhibit enzymes by binding very tightly to the Active Sites of enzymes; Sometoxic, naturally occurring and manufactured compounds are irreversible enzyme inhibitors; Examples: Penicillin inhibits Trans-peptidase needed in development of bacterial membrane; It prevents normal growth of bacteria; Aspirin inhibits Cyclooxygenaserequired for synthesis of Eicosanoids; Allopurinol inhibits Xanthine Oxidase required for the degradation of Purines 52  Enzyme Inhibition Many drugs are enzyme inhibitors Drug target use Aricept acetylcholinesterase Alzheimer disease Aspirin COX-1, COX-2 inflammation L-738,317 HIV protease HIV Lovastatin HMG CoA reductase high cholesterol Methotrexate dihydrofolate reductase cancer Penicillin bacterial transpeptidase antibiotic Caffeine cAMP phosphodiesterase stimulant 81 Dr. Salman Ashraf Chem 361– Biochemistry 82  Irreversible inhibition Forms stable covalent bond with enzyme inactivating it Nerve gas, is an irreversible inhibitor of the enzyme, acetylcholinesterase (AChE). When inactivated, the enzyme (AChE) can not transmit nerve impulses, thus leading to paralysis Death occurs by lung failure 83  Irreversible inhibition (example) Acetylcholinesterase (AChE). 84  Irreversible inhibition – DFP is an irreversible inhibitor of AChE DFP (An organophosphate) – A nerve gas Acetyl- cholinesterase (AChE). 85 Giveexamplesof enzymesthat are usedin diagnosis AcidPhosphatase:Tumor Marker in cancer of Prostate; Alanine Aminotransferase (ALT):used in LFT; Aspartate Aminotransferase (AST):use in LFT,Cardiac Function, Myocardial damage; Alkaline Phosphatase(ALP):use in LFT,Cholestatic liver disease; a marker of Osteoblast activity in bone disease; Amylase:Indicator of cell damage in acute Pancreatitis; Creatine Kinase(CK-MB): use in Myocardial damage; 86  Gamma-Glutamyl Transpeptidase(GGT): used in LFT,Hepato-Biliary damage and Alcoholism; LactateDehydrogenase(LDH): Muscle damage Cholinesterase involved in impulse transmission at neuromuscular and synaptic junctions, and PlasmaCholinesterase(Pseudo-Cholinesterase): It is involved in hydrolysis of Succinylcholine, a muscle-relaxant used in Anesthesia; Canbe used in diagnosis of poisoning with Pesticides, Insecticides, Organo-Phosphorus compounds; 87