Chapter 6 - Enzymes PDF

Enzymes Classes of Enzymes Nomenclature and Classification Enzymes are often classified by placing them in categories according to the reactions that they catalyze: 1. 2. 3. 4. 5. 6. Oxidoreductase Transferase Hydrolase Lyase Isomerase Ligase Oxidoreductases Catalyze oxidation-reduction reactions of...

Enzymes Classes of Enzymes Nomenclature and Classification Enzymes are often classified by placing them in categories according to the reactions that they catalyze: 1. 2. 3. 4. 5. 6. Oxidoreductase Transferase Hydrolase Lyase Isomerase Ligase Oxidoreductases Catalyze oxidation-reduction reactions of substrate molecules, most commonly addition or removal of O or H Require coenzymes that are reduced or oxidized as the substrate is oxidized or reduced Ex: alcohol dehydrogenase Transferases Catalyze the transfer of a groups from one molecule to another – Transaminases transfer an amino group between substrates – Kinases transfer a phosphate group from ATP to give ADP and a phophorylated product Hydrolases Catalyze the hydrolysis of substrates, i.e. the breaking of bonds with addition of water Important for digestion – Proteins are broken down to single amino acids – Carbohydrates are broken down to simple sugars Isomerases Catalyze the isomerization (rearrangement of atoms) of a substrate in reactions One substrate, one product Ex: triose phosphate isomerase Lyases Catalyze the addition of a molecules such as H2O, CO2, or NH3 to a double bond or the reverse reaction in which a molecule is eliminated to leave a double bond Ex: fumarase Ligases Catalyze the bonding together of two substrate molecules Not favorable, require simultaneous release of energy by hydrolysis reaction (i.e. conversion of ATP to ADP) Involved in the synthesis of proteins and DNA Classification of Enzymes 1. Oxidoreductase: O CH3 -C- COO Pyruvate - + NADH + H + lactate dehydrogenase OH + CH3 -CH-COO - + NAD Lactate 2. Transferase: COOCH2 CH- NH3 + + COO- COOAspartate amino C= O transferase CH2 COOCH2 C= O CH2 COO- + COOAspartate -Ketoglutarate COOC-N H3 + CH2 CH2 COO- Oxalosuccinate Glutamate 3. Hydrolase: O CH3 -C- OCH 2 CH 2 N( CH3 ) 2 + H2 O Acetylcholinesterease Acetylcholine O CH3 -C- O- HOCH2 CH2 N( CH3 ) 2 Acetate Choline Classification of Enzymes 4. Lyase: COOCH2 C-COO - + H O 2 CH COOcis- Aconitate COOCH2 H C-COO - Aconitase HO C-H COOIsocitrate 5. Isomerase: CH2 OPO 3 2 Phosphohexose O isomerase OH OH HO OH  - D- Glucose-6-phosphate 6. Ligase: CH2 OPO 3 2 O H HO H H HO CH2 OH OH  - D-Fructose-6-phosphate ATP + L-tyrosine + t-RNA Tyrosine-tRNA synthetase L-tyrosyl-tDNA + AMP + PP i Reaction Coordinate Diagram Comparing Enzyme-Catalyzed and Uncatalyzed Reactions Any reaction may have several steps, involving the formation and decay of transient chemical species called reaction intermediates. When several steps occur in a reaction, the overall rate is determined by the step (or steps) with the highest activation energy; this is called the rate-limiting step. GM = difference between transition state energies of uncatalyzed and catalyzed reactions Models for the binding of a substrate to an enzyme 1) The lock-and-key model Models for the binding of a substrate to an enzyme 2) The induced-fit model General mode of catalysis in enzymatic reactions: Effect of Substrate Concentration on Enzyme Activity Effect of Enzyme Concentration on Enzyme Activity Effect of Temperature on Enzyme Activity Effect of temperature and pH on enzyme activity Amino acid groups involved in catalytic mechanisms Chymotrypsin PG layer Inner membrane Outer membrane PG layer Inner membrane http://pathmicro.med.sc.edu/fox/cell_envelope.htm Transpeptidase is a bacterial enzyme Peptidoglycan is one of the few places in nature where D-amino acid residues are found. The active-site Ser attacks the carbonyl of the peptide bond between the two D-Ala residues, creating a covalent ester linkage between the substrate and the enzyme with release of the terminal D-Ala residue. It is the transpeptidase reaction that is inhibited by penicillin and related compounds, all of which mimic one conformation of the D-Ala to D-Ala segment of the peptidoglycan precursor. Transpeptidase Inhibition by β-Lactam Antibiotics β-Lactam antibiotics feature a five-membered thiazolidine ring fused to a four-membered β-lactam ring. The latter ring is strained and includes an amide moiety that plays a critical role in the inactivation of peptidoglycan synthesis. The R group varies in different penicillins. Penicillin G was the first to be isolated and remains one of the most effective, but it is degraded by stomach acid and must be administered by injection. Transpeptidase Inhibition by βLactam Antibiotics Attack on the amide moiety of the βlactam ring by a transpeptidase active-site Ser results in a covalent acylenzyme product. This is hydrolyzed so slowly that adduct formation is practically irreversible, and the transpeptidase is inactivated. β-Lactamases (bacterial) and β-lactamase inhibition β-Lactamases promote cleavage of the β-lactam ring in β-lactam antibiotics, inactivating them. Human use of penicillin and its derivatives has led to the evolution of strains of pathogenic bacteria that express β-Lactamases, enzymes that cleave β-Lactam antibiotics, rendering them inactive. The bacteria thereby become resistant to the antibiotics. β-Lactamases and β-lactamase inhibition The genes for these enzymes have spread rapidly through bacterial populations under the selective pressure imposed by the use (and often overuse) of βLactam antibiotics. Human medicine responded with the development of compounds such as clavulanic acid, which irreversibly inactivates the β-Lactamases. β-Lactamases and βlactamase Inhibition Clavulanic acid mimics the structure of a β-Lactam antibiotic and forms a covalent adduct with a Ser in the β-Lactamase active site. β-Lactam antibiotics (e.g. penicillin) β-lactamase (bacterial) Calvulanic acid Bind to transpeptidase (bacterial) Bacterial cell wall peptidoglycan cross-links formation a β-lactamase inhibitor Penicillin is a natural product—produced from Penicillium chrysogenum Enzyme Kinetics Rate of disappearance of A = − Δ  A  /Δt Rate of disappearance of B = − Δ  B /Δt Rate of appearance of P = Δ  P  /Δt Enzyme Kinetics (continued 1) Rate = −  A  t = −  B = t  P t – Negative sign indicates that A and B are used up in the reaction, while P is produced Rate   A   B f Rate = k  A   B f g g – k - Proportionality constant called the rate constant – f and g - Small whole numbers that are experimentally determined Enzyme Kinetics (continued 2) Overall order of a reaction is the sum total of all exponents – Rate of the reaction A → P is given by the 1 following equation: Rate = k  A  This reaction is first order with respect to A and first order overall – First order: Description of a reaction whose rate depends on the first power of the concentration of a single reactant Enzyme Kinetics (continued 3) Consider the reaction A + B → C + D whose rate equation is given by the 1 1 following expression: Rate = k  A   B – Where k is the rate constant, exponent for [A] is 1, and exponent for [B] is 1 – Reaction is said to be first order with respect to A, first order with respect to B, and second order overall Second order: Description of a reaction whose rate depends on the product of the concentrations of two reactants Second-Order Reactions Example Consider the reaction of glycogenn with inorganic phosphate, Pi – Reaction rate depends on concentrations of both reactants Glycogen n + Pi → Glucose-1-phosphate + Glycogen n−1 Rate = k  Glycogen   Pi  = k  Glycogen  Pi  1 1 – Reaction is first order with respect to glycogen, first order with respect to phosphate, and second order overall Zero-Order Reactions Reaction that proceeds at a constant rate, independent of the concentration of reactant For the reaction A → B Rate = k  A  = k 0 Rate depends on the presence of catalysts – Enzyme-catalyzed reactions exhibit zeroorder kinetics when the reactant concentrations are so high that the enzyme is completely saturated with reactant molecules Enzyme Kinetics Rate (Velocity) of an Enzymatic Reaction Initial rate, or initial velocity (Vinit or V0), and observed kinetics depend on [S] – [E] is constant At infinite [S], the reaction would proceed at maximum velocity (Vmax) Initial Velocities of Enzyme-Catalyzed Reactions S→P The Michaelis-Menten approach to enzyme kinetics Devised in 1913 by Leonor Michaelis and Maud Menten. Has undergone modifications but is still the basic model for non-allosteric enzymes. For the typical reaction, S P the mechanism for an enzyme catalyzed reaction can be summarized in the form: k1 k2 E+S ES E+P k-1 Assumption: product is not converted to substrate to any appreciable extent. k1 is the rate constant for the formation of the enzyme substrate complex, ES, from E and S. k-1 is the rate constant for the reverse reaction; dissociation of ES to free E and S. k2 is the rate constant for the conversion of the ES complex to product, P and the subsequent release of product from the enzyme, E (rate determining step). Michaelis–Menten Model KM - Inverse measure of the affinity of the enzyme for the substrate – Lower the KM, the higher the affinity Rate of formation of the ES is given by the following equation: Rate of formation = Δ  ES = k1  E S Δt – Δ[ES]/Δt - Change in concentration of the complex during a given time – k1 - Rate constant for the formation of the complex Michaelis–Menten Model (continued 1) Rate of the disappearance of the ES complex is given by the following equation: −Δ  ES Rate of breakdown = = k−1  ES + k2  ES Δt – –Δ[ES]/Δt Negative sign denotes that the concentration of the complex decreases as it breaks down – k–1 - Rate constant for the dissociation of the ES complex to regenerate enzyme and substrate – k2 - Rate constant for the reaction of the complex to give product and enzyme Michaelis–Menten Model (continued 2) Steady state: Condition in which the [ES] remains constant in spite of continuous turnover – Reached when the rate of formation of the ES equals the rate of its breakdown Δ  ES Δt = −Δ  ES Δt [S] is greater than [E] k1  E S = k−1  ES + k2  ES Michaelis–Menten Model (continued 3) When the steady state is reached, the concentration of free enzyme, [E], is given by the following reaction:  E  =  E  T −  ES – Where [E]T is the total concentration of the enzyme Substituting for the concentration of free enzyme, [E], gives: k1 ( E T −  ES) S = k−1  ES + k2  ES Michaelis–Menten Model (continued 4) Collecting all rate constants for the individual reactions gives: ( E T −  ES) S = k−1 + k2 = K M ES k   1 – KM is called the Michaelis constant In the initial stages of the reaction, formation of product depends only on the rate of breakdown of ES Vinit = k2  ES = k2  E T S K M + S (equation 1) Michaelis–Menten Model (continued 5) If substrate concentration is so large that the enzyme is saturated with substrate [ES] =  E T Vinit = Vmax = k2  E T – Substituting k2[E]T = Vmax into equation 1 gives the Michaelis–Menten equation Vinit = Vmax S K M + S Graphical Determination of Vmax and KM Constant rate when the enzyme is saturated with substrate is the Vmax for the enzyme V = Vmax S K M + S When [S] = KM: V = Vmax S S + S Vmax V = 2 Linearizing the Michaelis-Menten equation Lineweaver–Burk double reciprocal plot of enzyme kinetics The equation for a hyperbola can be manipulated into an equation for a straight line by plotting the reciprocal of reaction velocity, 1/V, vs. the reciprocal of the [S], 1/[S]. --Difficult to estimate Vmax and Km from a hyperbolic curve but convenient with a straight line. --Equation y = mx + b, where 1/V takes the place of y and 1/[S] is the x. The slope of the line, m, is Km/Vmax, and the intercept, b, is 1/Vmax. Inter 0.02 0.01 0.05 Inter -4 -2 -10 Km Vmax Inter Km -4 0.25 -2 0.50 -10 0.10 Inter Vmax 0.02 50 0.01 100 0.05 20 Inhibitors Interfere with enzyme action and slow reaction rate; effectiveness measured by KI (dissociation constant for E-I complex; lower KI corresponds to tighter binding inhibitor); alter apparent Km and Vmax. Two broad categories: 1) Reversible inhibitor- binds weakly to enzyme via non-covalent forces and later released, leaving original enzyme intact; can “equilibrate off.” 2) Irreversible inhibitor- reacts with the enzyme, often forming covalent bond, to produce a protein that is inactive; original enzyme cannot be regenerated. Reversible Inhibitors Two major classes of reversible inhibitors can be distinguished on the basis of the sites on the enzyme to which they bind. 1) Competitive Inhibitors -- very similar in chemical structure to substrate. -- bind at active site (E) and block access of substrate, preventing its binding. -- compete with substrate for active site. -- can be overcome by sufficiently high [S]. Kinetics of competitive inhibition ❖ Vmax unchanged relative to uninhibited reaction; can still achieve Vmax when [S]>>[I]. Lines therefore intersect on y-axis. ❖ Km increases by [1+ ([I]/KI)] (higher S required to achieve same rate); increasing inhibition leads to increasing slope. Reversible Inhibitors 2) Noncompetitive or mixed inhibitors --bind to enzyme at site distinct from active site. --leads to conformational change in enzyme structure, especially around active site. --substrate may still bind to active site, but enzyme cannot catalyze reaction. -- increasing [S] cannot overcome noncompetitive inhibition because substrate and inhibitor are not competing for same site; conformational alteration at active site still present no matter how much S. Fig. 6-11c, p.146 Noncompetitive Inhibition Form of enzyme inactivation in which a substance binds to a site other than the active site but distorts the active site to inhibit reaction Several equilibria are involved Involves two distinct binding sites, one for the substrate and one for the inhibitor Figure 6.17 - Nature of Substrate and Inhibitor Binding in Noncompetitive Inhibition Identifying a Noncompetitive Inhibitor Inhibitor does not interfere with substrate binding – Value of Vmax decreases, and value of KM remains the same Increasing substrate concentration cannot overcome noncompetitive inhibition – Inhibitor and substrate are not competing for the same site Identifying a Noncompetitive Inhibitor (continued) In presence of a noncompetitive inhibitor, I, maximum velocity of the reaction, Vimax, has the following form: I max V Vmax = 1 +  I  /K I – KI - Dissociation constant for the enzyme– inhibitor complex, EI Lineweaver–Burk Plot for Noncompetitive Inhibition Slope and y intercept change x intercept remains unchanged No inhibition KM 1 1 1 = × + V Vmax  S  Vmax y = m × x + b Noncompetitive inhibition I  I    KM  1 1 1  = + 1 + × 1 +  V Vmax  K I   S  Vmax  KI  y = m × x + b Figure 6.18 - Lineweaver–Burk Plot of Enzyme Kinetics for Noncompetitive Inhibition Example 6.2 Sucrose (common table sugar) is hydrolyzed to glucose and fructose in a classic experiment in kinetics – Reaction is catalyzed by the enzyme invertase – Using the following data, determine, by the Lineweaver–Burk method, whether the inhibition of this reaction by 2 M urea is competitive or noncompetitive Example 6.2 - Solution Plot the data with the reciprocal of the sucrose concentration on the x-axis and the reciprocals of the two reaction velocities on the y-axis as shown in Figure 6.19 – Note that the two plots have different slopes and different y intercepts, typical of noncompetitive inhibition – Note the same intercept on the negative xaxis, which gives –1/KM Figure 6.19 - Lineweaver–Burk Plot of the Data Mixed Noncompetitive Inhibition Similar to noncompetitive inhibition but the binding of I does affect the binding of S and vice versa – Dissociation constants KI and K′I are not identical Reversible Inhibitors Uncompetitive inhibitors and their kinetics --bind only to ES complex. --both Vmax and Km altered; parallel lines. --no effect on E-S binding. --often multi-substrate enzymes. Kinetics of mixed inhibition ❖ Vmax decreases by [1+ ([I]/KI)]; altered enzyme can never effectively achieve same Vmax. ❖ Km relatively unchanged (inhibitor does not affect binding of S, or affinity); lines intersect near x-axis, to left of y-axis. Irreversible Inhibitors ❖ Changes Km, Vmax or both ❖Examples are nerve gases and toxins ❖Also used to label the active site ❖Mechanismbased or suicide inhibitors (drugs) fall in this class Enzyme Regulation Enzymes are regulated in five different ways: – Feedback control – Allosteric control – Inhibition – Covalent modification – Genetic control Enzyme Regulation: Feedback Control Biochemical reaction pathways are a series of reactions subject to feedback control Feedback control is the regulation of an enzyme’s activity by the product of a reaction later in the pathway – The result of the process feeds information back to affect the beginning of the process Enzyme Regulation: Feedback Control Let’s consider the following series of reactions: A Enzyme 1 B Enzyme 2 C Enzyme 3 D Enzyme Regulation: Allosteric Control Allosteric control – binding of a molecule at one site on a protein affects the binding of another molecule at a different site From the Greek allos (other) and steros (space) Allosteric enzyme – activity is controlled by binding of activator/inhibitor at a location other than the active site – Binding changes the shape of the active site Enzyme Regulation: Inhibition Reversible or irreversible Competitive, uncompetitive, or mixed Enzyme Regulation: Reversible Uncompetitive Inhibition Inhibitor does not compete with the substrate for the active site Cannot bind to enzyme alone (binds to enzyme-substrate complex) Enzyme Regulation: Reversible Competitive Inhibition Inhibitor competes with the substrate for binding to the active site Binds reversible, no reaction Prevents substrate from entering the active site Enzyme Regulation: Covalent Modification Removal of a portion of an enzyme or addition of a group Zymogen, aka “proenzyme” – becomes an active enzyme after a chemical change Ex: pepsinogen (portion removed) Ex: glycogen phosphorylase (group added) Enzyme Regulation: Genetic Control Synthesis of proteins is regulated by genes Mechanisms controlled by hormones can accelerate or decelerate enzyme synthesis (we’ll talk about this further in Chapter 28)