ENZYMOLOGY Prof. Dr. Rasha El Sayed Al-Azhar University Enzyme Kinetics • Introduction to Enzyme Kinetics • Rate of a reaction • Order of a reaction • Michaelis-Menten equation ENZYMES KINETICS • Enzyme kinetics is the study of the rate of chemical reactions (velocity) that are catalyzed by enzymes. • Velocity (v) is assessed by the rate of change of substrate to product per unit time. • In enzyme kinetics, the reaction rate is measured and the effects of variation in the conditions of the reaction are investigated. ENZYMES KINETICS Importance 1-Drug Discovery and Development: Kinetic data help determine how effectively a drug inhibits a target enzyme. 2-Industrial Biocatalysis: It optimizes bioreaction performance, enabling more efficient production processes. 3-Metabolic Engineering: It is used to understand reaction fluxes and how organisms adapt to environmental changes. 4-Understanding Disease: It aids in studying enzyme dysfunction related to diseases ENZYMES KINETICS Transition state (Ea) Free energy Vary fom reaction to other The free energy of a chemical reaction is the amount of energy released in the conversion of reactants to products under standard conditions. ENZYMES KINETICS • All chemical reactions have an activation energy (Ea). • The activation energy is the minimum amount of energy needed for a reaction to occur. • Enzymes lower the Ea of a particular reaction (do this because they have a high affinity for a transition state). E + S ES • Enzymes assist in the reaction, so less energy is required. This means the reaction can occur more easily. This speeds up the rate of the reaction as it allows the product to be formed faster. ENZYMES KINETICS • An enzyme has a high affinity for the transition state (even higher than for its substrate). Therefore, when the substrate binds, it is quickly forced into the transition state (It is a state that exists between the substrate and the product). The enzyme is said to facilitate the formation of the transition state. • The transition state has a high energy, making it very unstable. It can only exist transiently. The transition state spontaneously turns into the more stable product with lower energy. The enzyme has a low affinity for the product and so the product is released. • Then, the rate of the enzymatic reaction is directly proportional to the concentration of ES. ENZYMES KINETICS The rate of the enzymatic reaction is directly proportional to the concentration of ES. • The concentration of ES changes as the reaction progresses. • Therefore, the rate of product formation also changes over time. • When the reaction reaches equilibrium (steady state) the concentration of ES (and therefore the rate) remains relatively constant. ENZYMES KINETICS • Reaction Kinetics • When an enzyme is added to a lot of substrate, the reaction occurs in three stages with distinct kinetics: of ES The pre-steady state phase is very short as equilibrium is reached within microseconds. If you measure the rate in the first few seconds of a reaction (V0), you are actually measuring the steady state. This is the rate used in Michaelis-Menten Kinetics. Effect of [S] on enzymatic reactions Increase in the substrate concentration gradually increases the velocity of enzyme reaction within the limited range of substrate levels. A rectangular hyperbola is obtained when velocity is plotted against the substrate concentration. Three distinct phases of the reaction are observed in the graph (A-linear; B-curve; Calmost unchanged). Maximum activity All enzymes combined with S enzyme activity increases FIG. Effect of substrate concentration on enzyme velocity (A- linear; B-curve; C-almost unchanged). Effect of [S] on enzymatic reactions FIG. Effect of substrate concentration on enzyme velocity (A- linear; B-curve; C-almost unchanged). Effect of [S] on enzymatic reactions Vmax = the maximum rate (velocity) of reaction when all enzyme active sites are saturated with substrate (all of the enzyme is bound to substrate; i.e., (enzyme is saturated with S); unit for Vmax is moles/time Km = the substrate concentration that gives half maximal velocity. [S] when v is equal to ½ Vmax , Unit for Km is concentration (moles/L = M) FIG. Effect of substrate concentration on enzyme velocity (A- linear; Bcurve; C-almost unchanged). Mixed/Order reaction: As increases, the reaction order transitions from firstorder to zeroorder Order of enzymatic reactions Zero-order reaction: When the [S] is much greater than Km, the rate of reaction is independent of substrate concentration. First-order reaction: the velocity of the reaction is proportional to the substrate concentration (i.e. [S] is less than Km) FIG. Effect of substrate concentration on enzyme velocity (A- linear; Bcurve; C-almost unchanged). Michaelis-Menten Kinetics Model Role of enzyme catalysis (V0): number of moles of product / second • Michaelis-Menten explained kinetic characteristics E = Enzyme S = Substrate P = Product ES = Enzyme-Substrate complex k1 = rate constant for the association of E with S to form ES k2 = rate constant for the dissociation of ES to E and S k3 = rate constant for the conversion of ES to E and the product Michaelis constant is given by the formula: Michaelis-Menten Kinetics Model The equation concerns the steady state of an enzymatic reaction with one substrate, is given by: where v = Measured velocity, Vmax = Maximum velocity, S = Substrate concentration, Km = Michaelis – Menten constant. Let us assume that the measured velocity (v) is equal to ½ Vmax. Then the equation may be substituted as follows: Michaelis-Menten Kinetics Model The equation concerns the steady state of an enzymatic reaction with one substrate, and is given by: Where, Vmax = Maximum velocity, S = Substrate concentration, Km = Michaelis – Menten constant. Michaelis-Menten Kinetics Model Km –Km is a measure of the affinity an enzyme has for its substrate, as a lower Km means that less of the substrate is required to reach half of Vmax. Km is inversely proportional to the affinity of the substrate to the enzyme (i.e., the higher the Km, the lower the affinity; The lower the Km, the higher the affinity Michaelis-Menten Kinetics Model Km – 1- It is a constant and characteristic feature of a given enzyme (comparable to a thumb impression or signature). 2- It is a representative for measuring the strength of ES complex. a. Small Km: A numerically small (low) Km reflects a high affinity of the enzyme for substrate, because a low concentration of substrate is needed to half-saturate the enzyme, that is, to reach a velocity that is 1⁄2Vmax. b. Large Km: A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration of substrate is needed to halfsaturate the enzyme. Km is inversely proportional to the affinity of the substrate to the enzyme (i.e., higher the Km, lower the affinity; lower the Km, higher the affinity
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