007-ENZYME KINETICS--1.pdf

Enzyme kinetics The Definition and Characteristics of Enzymes Enzymes are catalysts that increase the rate of a reaction without being changed themselves. Characters:  a protein  Catalyst  effects rate of reaction and not the equilibrium  unchanged at the end of reaction  effective in s...

Enzyme kinetics The Definition and Characteristics of Enzymes Enzymes are catalysts that increase the rate of a reaction without being changed themselves. Characters:  a protein  Catalyst  effects rate of reaction and not the equilibrium  unchanged at the end of reaction  effective in smaller quantities  efficient and specific  reaction can be reversed  activities affected by surroundings  may need helpers – cofactors/coenzymes  involve in multiple steps of biochemical pathways Active site of Enzyme: The active site is the region of the enzyme that binds the substrate, to form an enzyme–substrate complex, and transforms it into product (Binding site). The active site is a three-dimensional entity, often a cleft or crevice on the surface of the protein, in which the substrate is bound by multiple weak interactions (non- covalent bond). Two models have been proposed to explain how an enzyme binds its substrate: the lock-and-key model and the induced-fit model. Mechanism of enzyme action: A. Activation Energy: 1. All the reactions that proceed from initial substrates (initial state) to products (final state) consume energy. This is called free energy of the reaction. 2. However the substrates do not become products directly, but must be energized (absorb energy) to reach an activated or transition state. This energy is called activation energy. 3. At transition state, there is a high probability that a chemical bond will be made or broken to form the product. 4. The definition of activation energy: is the amount of energy required to raise all the molecules in one mole of a substance to the transition state. 5. The effect of enzymes: is to decrease the energy of activation. B. Active site: 1. The specificity of an enzyme is determined by: a) The functional group of the substrate (or product). b) The functional group of the enzyme and its cofactors. c) The physical proximity of these various functional groups: 1) During the enzyme action, there is a temporary combination between the enzyme and its substrate forming enzyme-substrate complex. This occurs at active site of enzyme. 2) This is followed by dissociation of this complex into enzyme again and products.  Charged Stabilized Interactions Nonpolar Polar Have a very Specific 3-D Shape With a Specific Arrangement of Functional Groups Flexible OH HO HO + Enzymes act as a Specific Platform ENZYMES: Bind ONLY specific things HO Bind them ONLY in a OH OH Specific 3-D Orientation OH HO HO - + SPECIFICITY is the Key to Enzyme Action Enzyme kinetics: It is the study of the velocity (rate) of reactions catalyzed by enzymes. A. Velocity of reaction: It is the increase in the concentration of product (Or decrease in concentration of substrate) with time. B. Initial velocity: 1. If an enzyme is incubated with its substrate and the appearance of the product with time is recorded on a graph, the resulting line will have the hyperbolic shape as in the figure: 2. The rate (velocity) of the reaction, which corresponds to the slope of this curve, is initially constant but gradually decreases. 3. The decline in the rate of the reaction may be due to: a) Depletion of the substrate, b) Inhibition of the enzyme by its product. c) Denaturation of the enzyme. 4. Initial velocity (VI) of the reaction: It Is the Initial portion of the reaction where the Increase In the concentration of the product is correlated constantly with time. For this reason, only the initial velocity (Vi) is used in calculating the kinetic parameters of the reaction. 5. The units of velocity: Is concentration of the product per unit time, e.g. micromoles I minute. C. Factors affecting enzyme activity: 1. Concentration of enzyme: The Initial velocity of a reaction is directly proportional to the amount of the enzyme present, provided that all other conditions remain constant. (Note: when the amount of enzyme in a reaction is doubled; the amount of substrate converted to product is doubled. Also when the amount of enzyme is tripled, the amount of substrate converted to product is tripled and so on). 2. Concentration of substrate: The initial velocity of a reaction is directly proportional to the amount of substrate present till it reaches a maximum point known as maximum velocity (Vmax), where any further Increase in the amount of substrate causes no increase in substrate concentration the velocity of the reaction. This is true If all other conditions especially enzyme concentration remain constant. Factors Affecting Enzyme Action Different enzymes show different response to changes in substrate concentration, temperature, and pH Reaction Rate (velocity of a reaction): Reaction rate is the number of substrate molecules converted to product per unit time and is usually expressed as µmoles product formed per minute The rate of reaction (enzyme catalyzed) increases with substrate concentration until a maximal velocity (Vmax), the plateau reflects the saturation with a substrate of all available binding sites on the enzyme Maximum activity  Increasing substrate concentration increases the rate of Reaction Rate reaction (enzyme concentration is constant)  Maximum activity reached when all of enzyme combines with substrate substrate concentration Most of enzymes show Vmax Maximum activity hyperbolic dependence of Reaction Rate velocity on substrate concentration Vmax/2 Km substrate concentration Factors Affecting Enzyme Action: Temperature Little activity at low temperature Rate increases with temperature (the velocity increased with Tem until a peak due to the increased number of molecules having sufficient energy to pass over the energy barrier and form product) Most active at optimum temperatures (usually 37°C in humans) Activity lost with denaturation at high temperatures decrease the velocity Optimum temperature Reaction Rate Low High Temperature Factors Affecting Enzyme Action: pH pH affect the ionization of the amino acids in the active site. R groups of amino acids in the active site should have proper charge to bind with the substrate the ionization or unionization is affected by the pH Maximum activity at optimum pH Tertiary structure of enzyme is correct Most lose activity in low or high pH, extremes of pH can also lead to denaturation of the enzyme because the structure of active protein depends on the ionic character of the amino acid Narrow range of activity, and Reaction Rate the pH optimum varies for different enzymes Optimum pH 3 5 7 9 11 pH Each enzyme has its optimum pH Rate of Reaction -The substrate molecules should have sufficient energy to overcome energy barrier to be converted into products. - only few molecules have energy to pass the energy gap  rate of reaction is determined by number of molecules that is converted into the product. - the lower the free energy of activation, the more molecules have sufficient energy to pass over the transition state the faster the rate of the reaction The enzymes don’t change the free energies of the reactants and products  don’t change the equilibrium of the reaction Michaelis-Menten Model Michalis and Menten proposed model that accounts for most features of enzyme- catalyzed reactions. In this model the enzyme reversibly binds its substrate to form ES complex that subsequently breaks down to product k1 k2 E+S ES E+P Where k-1 E: enzyme S: substrate ES: enzyme-substrate complex P: product k1 k-1 k2 are rate constants Michaelis-Menten Equation The Michaelis-Menten Equation describes how reaction velocity varies with substrate concentration  Conclusions about Michaelis-Menten kinetics Characteristics of Km:  The Km constant is a characteristics of an enzyme and particular substrate  Km reflects the affinity of the enzyme for a particular substrate  Km numerically equal to the concentration substrate at which the reaction velocity is equal to ½ Vmax.  Km dose not vary with concentration of the enzyme  low Km  high affinity; low [S] is needed to half-saturate the enzyme  large Km  low affinity; high [S] is needed to half-saturate the enzyme Relationship between the velocity to enzyme concentration: the rate of reaction is directly proportional to the enzyme concentration at all substrate conc. if the enzyme concentration is halved  the Vo is reduced to half Order of reaction: When [S] > Km, the V is constant and equal Vmax. The rate of reaction is independent on the [S] the reaction rate is Zero order  Double reciprocal of Michaelis-Menten equation Lineweaver-Burk Plot When V is plotted versus the [S], it is not always possible to determine the Vmax or Km from the graph because of gradual upward slope of the hyperbolic curve vo =Vmax [S] / (Km + [S]) (Michaelis-Menten Equation) Equation of straight line: y= ax + b (a = slope, b = y intercept Linear Transform: take reciprocal of each side of equation 1/vo = Km/Vmax 1/[S] + 1/Vmax y = a x + b  Lineweaver-Burk Double Reciprocal Plot 1/vo = Km/Vmax 1/[S] + 1/Vmax Lineweaver-Burk line allow us to determine the Vmax and Km simply The x intercept is -1/km The y intercept is 1/Vmax  Enzyme Inhibition Enzyme inhibitor: any substance that can reduce the velocity of an enzyme- catalyzed reaction and cause a loss of catalytic activity Inhibitors can be reversible or irreversible. - Reversible inhibitors bind to enzymes through a non-covalent bonds  Upon dilution the EI (enzyme-inhibitor) complex dissociate and recover the enzyme activity, may be competitive or noncompetitive - Irreversible inhibition occur when the inhibited enzyme can not recover its activity by dilution. The inhibitors form a covalent bonds with the active site enzyme or destruction of the protein structure of the enzyme Irreversible Inhibitors Competitive inhibitors This type of inhibition occurs when the inhibitors bind reversibly to the same site that the substrate normally occupy  compete the substrate for that site A competitive inhibitor Has a structure similar to substrate Occupies active site Competes with substrate for active site Has effect reversed by increasing substrate concentration Competitive inhibitors Vmax: the effect of the competitive inhibitors is reversed by increasing the [S]. At sufficient high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of the inhibitors. The y-intersect is unchanged Km: a competitive inhibitors increases the apparent Km for a given substrate in the presence of this inhibitors , more substrate is needed reach the Vmax. The x intersect is changed indicating that the apparent Km is increased Noncompetitive Inhibition Non competitive inhibition occurs when the inhibitor at a site distinct from the substrate site A noncompetitive inhibitor does not have a structure like substrate. Alter the shape of enzyme and active site Substrate cannot fit altered active site It binds either to the enzyme or to the ES complex No reaction occurs Effect is not reversed by adding substrate It may bind to free E or to ES. Once bound it will prevent P formation. And the affinity of the I to both E and ES is the same.  Non-competitive inhibitors decrease the Vmax, and can not be overcome by increasing the substrate concentration Non-competitive inhibitors don’t affect the Km, the enzyme show the same Km in the absence or the presence of the inhibitors Enzyme inhibitors could be used as drugs Different types of enzyme inhibitors I2 I1 NONCOMP I0 COMP  Enzyme inhibitor: harmful or beneficial?  Sarin – the nerve gas Action – inhibits acetylcholinesterase from hydrolyzing acetylcholine to acetate & choline Effect – acetylcholine gather at end of nerve, causing symptoms such as fuzzy eyesight, extreme sweating, loss of motor functions control & paralysis acetylcholinesterase – enzyme in the body which has an important function in nerve regulation and control  Penicillin – antibacterial agent Action – covalently attaches to bacterial glycoprotein peptidase active site, preventing peptidoglycan peptide bond cross-linking Effect – prevents cell wall synthesis; exposing bacterial cell to osmotic lysis; bacteria cannot reproduce glycoprotein peptidase – bacterial enzyme catalyzing cross-linking of peptidoglycan peptide bonds, the main cell wall polymer