Enzyme Kinetics PDF

Enzyme kinetics Kinetics is the study of reaction rates (velocities). Study of enzyme kinetics is useful for measuring – concentration of an enzyme in a mixture (by its catalytic activity), – its purity (specific activity), – its catalytic efficiency and/or specificity for different su...

Enzyme kinetics Kinetics is the study of reaction rates (velocities). Study of enzyme kinetics is useful for measuring – concentration of an enzyme in a mixture (by its catalytic activity), – its purity (specific activity), – its catalytic efficiency and/or specificity for different substrates – comparison of different forms of the same enzyme in different tissues or organisms, – effects of inhibitors (which can give information about catalytic mechanism, structure of active site, potential therapeutic agents...) 1 Enzyme Kinetics Equation 2  Initial Velocity (vo) and [S] The concentration of substrate [S] present will greatly influence the rate of product formation, termed the velocity (v) of a reaction. Studying the effects of [S] on the velocity of a reaction is complicated by the reversibility of enzyme reactions, e.g. conversion of product back to substrate. To overcome this problem, the use of initial velocity (vo) measurements are used. At the start of a reaction, [S] is in large excess of [P], thus the initial velocity of the reaction will be dependent on substrate concentration. When initial velocity is plotted against [S], a hyperbolic curve results, where Vmax represents the maximum reaction velocity. At this point in the reaction, if [S] >> E, all available enzyme is "saturated" with bound substrate, meaning only the ES complex is present. 3 Substrate Saturation of an Enzyme A. Low [S] B. 50% [S] or Km C. High, saturating [S] 4 Michaelis-Menten Curve Michaelis-Menten equation explains hyperbolic Vo vs. [S] curve: 1. At very low [S] ([S] > Km), Vo approaches Vmax (velocity independent of [S]) 5 Michaelis-Menten equation Dependence of velocity on [substrate] is described for many enzymes by the Michaelis-Menten equation: Km (the Michaelis constant) Kinetic parameters can be determined graphically by measuring velocity of enzyme-catalyzed reaction at different concentrations of substrate (Vo vs. [substrate]). 6 Michaelis-Menten equation Dependence of velocity on [substrate] is described for many enzymes by the Michaelis-Menten equation: Km (the Michaelis constant) Kinetic parameters can be determined graphically by measuring velocity of enzyme-catalyzed reaction at different concentrations of substrate (Vo vs. [substrate]). 7  Meaning of Km An important relationship that can be derived from the Michaelis-Menten equation is the following: If vo is set equal to 1/2 Vmax, then the relation Vmax /2 = Vmax[S]/Km + [S] can be simplied to Km + [S] = 2[S], or Km = [S]. This means that at one half of the maximal velocity, the substrate concentration at this velocity will be equal to the Km. In the simplest assumption, the rate of ES breakdown to product (k2) is the rate-determining step of the reaction, so k-1 >> k2 and Km = k-1/k1. This relation is also called a dissociation constant for the ES complex and can be used as a relative measure of the affinity of a substrate for an enzyme (identical to Kd). High KM = high dissociation = weak binding between E and S. Km = substrate concentration that gives Vo = 1/2 Vmax. 8  Lineweaver-Burk (double reciprocal plot) If the reciprocal (1/X) of the Michaelis-Menten equation is done, after algebraic simplification the following equation results: This relation is written in the format of the equation for a straight line, y = mx + b, where y = 1/vo, m (slope) = Km/Vmax, x = 1/[S] and the y-intercept, b = 1/Vmax. When this relation is plotted,the result is a straight line graph 9 Lineweaver-Burk (double reciprocal plot) (cont) 10 Uses of double reciprocal plot The x intercept value is equal to -1/Km. The biggest advantage to using the double reciprocal plot is a more accurate determination of Vmax, and hence Km. It is also useful in characterizing the effects of enzyme inhibitors and distinguishing between different enzyme mechanisms. 11 Enzyme inhibitors Whenever, an anzyme reaction is proceeding at a rate less than that expected for existing condtions of pH, temp., substrate and activator conce., the enzyme is said to be inhibited. Two forms of enzyme inhibitors may be encountred reversible and irreversible inhibition. 12 Enzyme Inhibitors Specific enzyme inhibitors regulate enzyme activity and help us understand mechanism of enzyme action. (Denaturing agents are not inhibitors) Reversible inhibitors form an EI complex that can be dissociated back to enzyme and free inhibitor. 3 groups based on their mechanism of action: competitive, non-competitive and uncompetitive.. Irreversible inhibitors form covalent or very tight permanent bonds with aa at the active site of the enzyme and render it inactive. 3 classes: groupspecific reagents, substrate analogs, suicide inhibitors 13 Reversible inhibition For reversible inhibitors, a term Ki can be determined. For competitive inhibitors, the following relation can be used: Km + I = Km (1 + [I] / Ki ) ; (where Km + I is the determined Km in the presence of [I]). Ki values are used for comparison of the different types of inhibitors. In general, the lower the Ki value, the tighter the binding, and hence the more effective an inhibitor is 14 Reversible inhibition 1. Competitive: The inhibitor’s action is proportional to its concentration. Resembles the substrate’s structure closely. Compete with substrate for binding to enzyme E + S = ES or E + I = EI. Both S and I cannot bind enzyme at the same time In presence of I, the equilibrium of E + S = ES is shifted to the left causing dissociation of ES. This can be reversed / corrected by increasing [S] Vmax is not changed, KM is increased by (1 + I/Ki) Eg: , antibacterial E + I sulfonamides, EI the anticancer agent methotrexate etc Reversible Enzyme inhibitor reaction complex 15 Competitive Inhibition Vmax - No change Km INCREASES - indicates a direct interaction 16 of the inhibitor in the active site  Reversible Non- Competitive Inhibition Non-competitive inhibitors binding site is distinct from substrate binding site. Can bind to free enzyme E and to ES E + I = EI, ES + I = ESI or EI + S = ESI Both EI and ESI are enzymatically inactive. The inhibitor binds to a site other that the substrate site, and is thus independent of the presence or absence of substrate. This action results in a conformational change in the protein that affects a catalytic step and hence decreases or eliminates enzyme activity (formation of P). Notice in the reciprocal plot, a non-competitive inhibitor does not affect the binding of the substrate (Km), but it does result in a decrease in Vmax. This can be explained by the fact that since inhibitor bound to an enzyme inactivates it, the more EI formed will lower [ES] and thus lower the overall rate of the reaction Vmax. Inhibition cannot be reversed by increasing [S] KM is not changed, Vmax is decreased. 17  Reversible Non- Competitive Inhibition Non-competitive inhibitors binding site is distinct from substrate binding site. Can bind to free enzyme E and to ES E + I = EI, ES + I = ESI or EI + S = ESI Both EI and ESI are enzymatically inactive. The inhibitor binds to a site other that the substrate site, and is thus independent of the presence or absence of substrate. This action results in a conformational change in the protein that affects a catalytic step and hence decreases or eliminates enzyme activity (formation of P). Notice in the reciprocal plot, a non-competitive inhibitor does not affect the binding of the substrate (Km), but it does result in a decrease in Vmax. This can be explained by the fact that since inhibitor bound to an enzyme inactivates it, the more EI formed will lower [ES] and thus lower the overall rate of the reaction Vmax. Inhibition cannot be reversed by increasing [S] KM is not changed, Vmax is decreased. 18  Non-Competitive Inhibition Vmax DECREASES - inhibitor affects rate of reaction by binding to site other than substrate active-site Km - No change 19 Uncompetitive reversible Inhibitors The inhibitor cannot bind to the enzyme directly, but can only bind to the enzyme-substrate complex. ES + I = ESI. Both Vmax and KM are decreased. 20 Irreversible inhibitors generally result in the destruction or modification of an essential amino acid required for enzyme activity. Frequently, this is due to some type of covalent link between enzyme and inhibitor. These types of inhibitors range from fairly simple, broadly reacting chemical modifying reagents (like iodoacetamide that reacts with cysteines) to complex inhibitors that interact specifically and irreversibly with active site amino acids. (termed suicide inhibitors). These inhibitors are designed to mimic the natural substrate in recognition and binding to an enzyme active site. Upon binding and some catalytic modification, a highly reactive inhibitor product is formed that binds irreversibly and inactivates the enzyme. Use of suicide inhibitors have proven to be very clinically effective 21 Examples of Irreversible inhibition Cyanide combines with the Iron in the enzymes cytochrome oxidase. Heavy metals, Ag+ or Hg+2, combine with –SH groups on enzymes. These can be removed by using a chelating agent such as EDTA. Alkylating agents: such as iodoacetamide 22 Diisopropyl Phosphofluoridate: Irreversible Acetylcholinesterase Inhibitor ( nerve gase Example) 23 Example of reversible competitive inhibition Succinate Fumarate + 2H++ 2e- Succinate dehydrogenase CH2COOH COOH CHCOOH CH2 CH2COOH CHCOOH COOH succinate(substrate) fumarate(product) Malonate (inhibitor) 24