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Juhi Ramchandani 10/02/12 Notes Biochemistry Chapter 13: Enzymes Kinetics and Specificity 1. Enzymes a. Biological Catalysts b. Highly specific (for a substrate) c. Binding takes place at active site (crevice on surface of enzyme) d. Substrate binds to enzyme (at active site); must bind for reaction to occur and for product synthesis e. Products synthesized are highly specific (no side reactions, like nonbiological catalysts) f. Catalytic power, specificity (selectivity for substrates and products), and regulation (of activity for integration and regulation of metabolism) g. Promote the creation of a transition state h. Decrease the activation energy of a reaction 2. Enzyme Nomenclature a. Sapling: The International Union of Biochemistry has suggested that enzymes be classified in one of six categories. i. Oxidoreductases catalyze oxidation-reduction reactions. ii. Transferases catalyze the transfer of functional groups. iii. Hydrolases catalyze hydrolysis of chemical bonds. iv. Isomerases catalyze isomerization of a substrate. v. Lyases catalyze a group elimination to form a double bond, or addition of a group to a double bond. vi. Ligases catalyze bond formation coupled to ATP hydrolysis to provide energy. b. Hydrolase i. Addition of a water molecule to a larger molecule, and hydrolase breaks larger molecule into two components (at carbonyl oxygen) to create carboxylic group on one end and protonated group on other molecule ii. Catalyzes hydrolysis reaction of large macromolecule into two smaller molecules iii. A number of different proteolytic enzymes (proteases) catalyze this reaction iv. Bidirectional? c. Isomerase i. Creates a product with the same formula, just different atom connectivity ii. Creates an isomer of the initial compound iii. From HW: 1. One carbon atom has been oxidized and another reduced 2. The substrate and product are therefore isomers

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3. This particular enzyme is very important in glycolysis, and is called triosephosphate isomerase d. Oxioreductase i. Reduces a molecule (ex. Carbonyl group to alcohol) by using NADH and acid (H+) ii. A product is NAD+ iii. The substrate is reduced by the addition of two hydrogen atoms while the coenzyme NADH is being oxidized iv. Redox reaction v. Bidirectional? 1. From HW: This particular enzyme is named in the reverse direction and is known as glycerol-3-phosphate dehydrogenase. It is an important enzyme involved in transferring electrons from the cytoplasm into mitochondria and in synthesis of phospholipids and triglyerides e. Lyase i. Catalyze group elimination ii. In other words, break up a macromolecule at the site of a double bond to induce a second double bond formation f. Ligase i. Join two molecules together by coupling them with ATP hydrolysis (ATP ADP + Pi) ii. Ex. Carboxylation reaction coupled with ATP hydrolysis iii. Energy for bond formation provided by ATP hydrolysis iv. In other words, cleaving ATP for bond formation g. Transferase i. Catalyze the transfer of functional groups [from one molecule to another] ii. Ex. Transfer of phosphate group from PEP to ADP (pyruvate kinase = transferase) 3. Cofactor a. Nonprotein components of a protein structure required by many enzymes to carry out their catalytic function (unable to rely solely on protein structure) b. Metal ions are cofactors that may be either tightly or loosely bound to an enzyme (Metal ions that are tightly bound to an enzyme are sometimes called prosthetic groups) c. Ex. Fe2+/3+, Cu2+, Zn2+, Mg2+, Mn2+, K+, Ni2+, Mo, Se 4. Coenzyme a. Cofactors that are organic molecules b. Nonprotein organic component of a protein structure c. Ex. Thiamine pyrophosphate (TPP), Flavin adenine dinucleotide (FAD), Nicotinamide adenine dinucleotide (NAD), Coenzyme A (CoA), Pyridoxal phosphate (PLP), 5-Deoxyadenosylcobalamin (vitamin B12), Biotin (biocytin), & Tetrahydrofolate (THF) d. NADH, biotin, and CoQ are organic, nonprotein components 5. Enzyme Kinetics

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a. Enzyme kinetics seeks to determine the maximum reaction velocity that enzymes can attain and the binding affinities for substrates and inhibitors b. Analysis of enzyme rates yields insights into enzyme mechanisms and metabolic pathways c. This information can be exploited to control and manipulate the course of metabolic events d. Kinetics cannot prove a reaction mechanism e. Kinetics can only rule out various alternative hypotheses because they dont fit the data f. In a general enzyme-catalyzed mechanism, the enzyme (E) and substrate (S) must first come in contact with each other (E S). The first step of the mechanism is the binding of the substrate and enzyme to form an enzymesubstrate complex (ES). The enzyme then catalyzes the conversion of substrate to product (P), and the product is released (E P). g. Pharmaceuticals/drugs: special inhibitors specifically targeted at a particular enzyme to overcome infection or alleviate illness h. Velocity/rate of reaction: amount of Product (P) formed or amount of Reactant (A) consumed over time; rate is proportional to [A] i. Rate constant: (k), with units per time (sec-1) j. Order: exponent of concentrations in rate equation k. Molecularity: number of molecules that must simultaneously react i. Unimolecular reaction: molecularity equals 1 (ex. Rate of decay of a radioactive isotope, or intramolecular rearrangement AP); first order reactions ii. Bimolecular reaction: molecularity equals 2 (second order reactions); 2 reactants must collide in order for reaction to occur, so velocity can be monitored by rate of disappearance of either reactant l. Transition state: energy necessary to achieve a reactive condition m. Free Energy of Activation: height of the energy barrier needed to overcome in order for reaction to proceed toward product synthesis (energy required to raise the average energy of 1 mol of reactant at a given temperature to the transitionstate energy) i. Decreasing it increases reaction rate 6. What Equations Define the Kinetics of Enzyme-Catalyzed Reactions? a. Plot of v (velocity) vs. reactant concentration yields a straight line with slope k i. At low concentrations of the substrate S, v is proportional to [S] (first order w/ respect to [S]) b. At high [S], v becomes virtually independent of [S] and approaches a maximal limit (0th order w/ respect to [S]) i. Saturation effect: rate is independent of substrate concentration 1. When v does not increase w/ [S] increase (substrate saturation curves) c. Substrate binds at the active site of the enzyme i. Conformation of the active site is structured to form a special pocket or cleft whose three-dimensional architecture is complementary to the structure of the substrate.

Ramchandani Ch 13 ii. Recognition thru structural complementarity! iii. Substrate binds to the enzyme through relatively weak forces H bonds, ionic bonds (salt bridges), and van der Waals interactions between sterically complementary clusters of atoms. d. Michaelis-Menten Equation

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i. Based on notion that Enzyme & Substrate reversibly associate to form an ES complex ii. Association/dissociation is assumed to be a rapid equilibrium iii. Ks = enzyme-substrate dissociation constant iv. Made several assumptions for theory 1. Assumes the formation of an enzyme-substrate complex 2. Assumes that the ES complex is in rapid equilibrium with free enzyme 3. Assumes that the breakdown of ES to form products is slower than 1) formation of ES and 2) breakdown of ES to re-form E and S v. Assuming CONSTANT [ES] (steady-state assumption) 1. ES is formed as rapidly from E + S as it disappears by its two possible fates: dissociation to regenerate E + S and reaction to form E + P. 2. Normally enzyme concentration is much lower than substrate (otherwise would be a reactant) 3. Assuming that there is a steady-state population of E-S complex b/c of low initial enzyme concentration 4. Taking a derivative of the [ES] gives 0, indicating a constant concentration (steady state concept reinforced) vi. Assuming velocity measurements made immediately following S addition & the total amount of enzyme is fixed gives the Michaelis constant Km: 1. (Km ~ Vmax/2)

2. -OR3. When [S] 0, v is linear w/ slope 1/Km 4. When [S] , v = Vmax e. Rate velocity i. Increases when additional substrate is added with low substrate concentration (enzyme is not saturated) ii. Is unchanged when: 1. Additional substrate is added when substrate concentration is high 2. Substrate is added when enzyme is saturated with substrate iii. Describes a rectangular hyperbola iv. Km and Vmax, once known explicitly, define the rate of the enzymecatalyzed reaction, provided:

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1. The reaction involves only one substrate, or if the reaction is multisubstrate, the concentration of only one substrate is varied while the concentrations of all other substrates are held constant. 2. The reaction ES E + P is irreversible, or the experiment is limited to observing only initial velocities where [P] = 0. 3. [S]0 > [ET] and [ET] is held constant. 4. All other variables that might influence the rate of the reaction (temperature, pH, ionic strength, and so on) are held constant. v. The "kinetic activator constant" 1. When Km is experimentally determined, it is a constant that is characteristic of the enzyme and the substrate under specific conditions 2. The lower the value of Km, the greater the affinity of the enzyme for ES complex formation 3. Michaelis constant: The rate of the forward reaction of the ES complex plus the rate of the reverse reaction from this complex divided by the rate of ES production 4. Km is a constant derived from rate constants 5. Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S 6. Small Km means tight binding; high Km means weak binding vi. The theoretical maximal velocity 1. Vmax is the maximum velocity a reaction can attain 2. Vmax is a constant 3. Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality 4. To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate 5. Vmax is asymptotically approached as substrate is increased vii. Turnover number (kcat): measure of the maximum catalytic activity of an enzyme 1. The number of substrate molecules converted to product per unit time is kcat 2. kcat is Vmax over total enzyme concentration (Et) 3. The number of substrate molecules converted into product per

enzyme molecule per unit time when the enzyme is saturated with substrate.4. A.k.a. molecular activity 5. Represents kinetic efficiency of an enzyme 6. The catalytic effects of an enzyme cannot exceed the diffusioncontrolled rate of combination of E and S to form ES (in , k1 sets the upper limit) a. Kcat/km is the second order rate constant b. 108 to 109 (Ms)-1 is the diffusion-controlled rate in aqueous solutions

Ramchandani Ch 13 c. Enzymes w/ diffusion rates close to this are said to have achieved catalytic perfection viii. Specific Activity of an enzyme: units of enzyme activity; enzyme units per mg protein 7. Line Plots can be derived from the Michaelis-Menten Equation a. Lineweaver-Burk Plot

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i. Double replacement plot ii. Slope: Km/Vmax iii. Y-intercept: 1/Vmax iv. X-intercept: -1/Km b. Hanes-Woolf Plot i. Found by multiplying Lineweaver-Burk equation by [S] ii. Best b/c has smaller, more consistent errors across plot & allows extrapolation iii. iv. Slope: 1/Vmax v. Y intercept: Km/Vmax vi. X intercept: -Km c. Allosteric enzymes (regulatory enzymes) disobey the kinetics portrayed by the Michaelis-Menten equation! 8. Enzymatic Activity is Strongly Influenced by pH a. Enzyme-substrate recognition and catalysis are greatly dependent on pH b. Enzymes have a variety of ionizable side chains that determine its secondary and tertiary structure and also affect events in the active site c. The substrate may also have ionizable groups d. Enzymes are usually active only over a limited range of pH e. The effects of pH may be due to effects on Km or Vmax or both i. Pepsin, catalase, papain, cholinesterase, trypsin, fumarase, ribonuclease, arginase 9. Response of Enzymatic Activity to Temperature is Complex a. Rates of enzyme-catalyzed reactions generally increase with increasing temperature b. However, at temperatures above 50to 60C, enzymes typically show a decline in activity c. Two effects here: i. Enzyme rate typically doubles in rate for ever 10 C as long as the enzyme is stable and active ii. At higher temperatures, the protein becomes unstable and denaturation occurs (higher orders of proteins are unstable at high temps) d. Enzymes of thermophilic prokaryotes retain full activity at temperatures above 850C 10. Inhibition of Enzyme Activity

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a. Enzymes may be inhibited reversibly or irreversibly b. Reversible Inhibition i. Interact with enzyme through noncovalent association/dissociation reactions ii. Reversible inhibitors may bind at the active site or at some other site iii. Competitive 1. Inhibitor binds non-covalently to active site (competes with substrate) 2. A sufficiently high substrate concentration can displace inhibitor from enzyme a. NO INHIBITION W/ HIGH [S]! b. Vmax is the same in the presence & absence of the inhibitor c. Km is INCREASED! (b/c inhibitor competes w/ substrate to bind to active site) 3. Inhibitor usually resembles substrate structurally 4. Ex. Malonate is inhibitor of Succinate 5. Ex. Substrate analogs & transition state analogs (b/c resemble substrate or transition state) a. Tamiflu is a transition state analog that reversibly binds to neuraminidase iv. Non-competitive 1. Inhibitor binds non-covalently at a site other than the active site 2. Interaction with inhibitor results in a change in the enzymes conformation, making it unable to bind to the substrate (changes conformation of active site?) 3. Vmax is LOWER b/c high [S] cannot displace effects c. Irreversible Inhibition i. Irreversible inhibitor binds covalently and tightly ii. Cause stable, covalent alterations iii. May bind to active site or to any other site on enzyme iv. Inhibitor may permanently modify an enzyme v. Consequence: decrease in active enzyme concentration vi. Ex. DIPF reacts w/ OH group on serine residue of chymotrypsins active site, adding the DIP group to it and permanently modifying it vii. Ex. Mercury can poison an enzyme viii. Penicillin is an irreversible suicide inhibitor 1. Penicillin is an irreversible inhibitor of the enzyme glycoprotein peptidease, which catalyzes an essential step in bacterial cell all synthesis 2. Penicillin tightly binds to enzymes active site why it is called irreversible (tight fit/binding that cannot be reversed) 11. Effects of Inhibitors on Enzyme Kinetics a. Competitive Inhibition i. Only affects slope of graph (Lineweaver-Burk plot) 1. V is less in the presence of the inhibitor

Ramchandani Ch 13 2. At a given [I], v decreases 3. When [S] increases, v=Vmax and is unaffected by inhibitor 4. X & Y intercepts are unaffected ii. Inhibitor binds only to free enzyme b. Pure Non-competitive Inhibition i. Interact w/ both E and ES ii. Changes X & Y intercepts iii. Smaller Vmax for non-competitive inhibitor b/c changes active site shape, preventing binding of substrate and decrease overall binding activity of the enzyme iv. Does NOT affect Km

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12. Enzymes Catalyzing Bimolecular Reactions a. Enzymes often catalyze reactions involving two (or more) substrates b. Bisubstrate reactions may be sequential or single-displacement reactions or double-displacement (ping-pong) reactions c. Single-displacement reactions can be of two distinct classes: i. 1. Random, where either substrate may bind first, followed by the other substrate ii. 2. Ordered, where a leading substrate binds first, followed by the other substrate d. In a single displacement bisubstrate mechanism, slope decreases with increase in concentration of second substrate e. Double Displacement involves shift of line (parallel lines) to the right (horizontal shift) with increase in second substrate concentration 13. How Enzymes can be so specific a. The Lock and key hypothesis was the first explanation for specificity b. The Induced fit hypothesis provides a more accurate description of specificity c. Induced fit favors formation of the transition state d. Specificity and reactivity are often linked. In the hexokinase reaction, binding of glucose in the active site induces a conformational change in the enzyme that causes the two domains of hexokinase to close around the substrate, creating the catalytic site e. Figure 13.24 A drawing, roughly to scale, of H2O, glycerol, glucose, and an idealized hexokinase molecule. Binding of glucose in the active site induces a conformational change in the enzyme that causes the two domains of hexokinase to close around the substrate, creating the catalytic site.