Enzymes and kinetics. Eva Samcová and Petr Tůma

Transcription

1 Enzymes and kinetics Eva Samcová and Petr Tůma

2 Termodynamics and kinetics Equilibrium state ΔG 0 = -RT lnk eq ΔG < 0 products predominate ΔG > 0 reactants predominate Rate of a chemical reaction Potential energy barrier activation energy

3 The rate of chemical reaction is measured Change in concentration of reacting substances per time unit reactant product Δ produkt v Δt Δ reaktant v Δt 1 Δ H2 v 3 Δt Δ N 2 v Δt 1 Δ NH v 2 Δt 3 3H 2 + N 2 2NH 3

4 The rate of chemical reaction depends on concentration A + B products Kinetic equation: v = k [A] [B] The rate of reaction is directly proportional to actual product of concentrations of reactants. Reaction order : first-order reaction, second-order reaction Collision Theory: A given reaction will be faster the more frequently the reactant particles collide. The number of active collisions between molecules A and B per second = [A] [B]

5 Activation energy and temperature v = k [A] [B] Rate constant k ~ exp(-e A /RT) exp(-e A /RT) Boltzman factor Boltzman factor = part of molecules in system with energy exceeding the activation energy (E A ) Effect of temperature: rate of chemical reaction doubles for every 10 C temperature rise (but not always)

6 Dependance on temperature v = k [A] [B] The rate constant k is independent of the concentrations of the species taking part in the reaction but depends on the temperature. Enzyme-catalyzed reactions may show more complex temperature dependence because raising the temperature may provoke conformational changes and even denaturation, degradation that lower effectiveness of the enzyme. Rate constant k ~ exp(-e A /RT) exp(-e A /RT) Boltzman factor

7 The Arrhenius equation The larger this ratio, the smaller the rate (hence the negative sign). This means that high temperature and low activation energy favor larger rate constant, and thus speed up the reaction rate. Because these terms occur in a exponent their effect on the rate are quite substantial.

8 Principle of Catalysis Catalysts are substances that increase the rate of chemical reaction without themselves being consumed in the process catalyst decreases activation energy 2 H 2 O 2 2 H 2 O + O 2 catalyst E A, kj mol -1 Without catalyst 75,4 Pt 48,6 Enzyme : catalase 23,0 Ni,Co CO H2 C3H8 H2O

9 General Properties of Enzymes Key Concepts Enzymes differ from ordinary chemical catalysts in reaction rate, reaction conditions, reaction specificity, and control. The unique physical and chemical properties of the active site limit an enzyme s activity to specific substrates and reactions. Some enzymes require metal ions or organic cofactors.

10 Enzymes Exhibit High Reaction Rates

11 Enzymes are Classified by Reaction Type

12 Classes of Enzymes

13 History and nomenclature of enzymes 1810, Gay-Lussac made an experiment with yeats alter saccharide to ethanol and CO 2 Fermentation From greek: en = in, zyme = yeats Historical names- pepsin, ptyalin substrate + -asa : ureasa, sacharasa Classification of enzymes X.X.X.X. class, subclass, sub-subclass, serial number of the enzyme in its sub-subclass lactate dehydrogenase 1. oxidoreductases 1.1. donor e - -CH-OH acceptor e - NAD + Reaction: Lactate + NAD + pyruvate + NADH

14 Chemical composition of enzymes holoenzyme = apoenzyme + cofactor Ribozymes (RNA) cofactors metal ion: Zn 2+ (ADH), Mn 2+ (arginasa), Mg 2+, Fe 2+, Cu 2+ coenzym: NAD, ubichinon, ATP, coenzym A prosthetic group: heme, FAD, lipoamide Carbonic anhydrase Phenylalanine hydroxylase

15 Cofactors Expand the Range of Enzymatic Reactions

16 Specificity of enzymes Substrate specificity absolute (urease) relative (group) glucose/fructose (hexokinase), ethanol/methanol (ADH) different affinity (K M ) Reaction specificity Reaction of amino acids deamination, decarboxylation, transamination Dependent on coenzymes and apoenzymes Stereospecificity D-monosaccharides, L-amino acids

17 Some Enzymes Catalyze Highly Stereospecific Reactions

18 Some Enzymes are More Permissive

19 Kinetics and Enzyme Kinetics Kinetics the main role of reaction kinetics is to measure the rates of chemical reactions and to deal with the factors which effect the rates. Enzyme Kinetics the dependence of the rate of an enzyme catalyzed reaction on the reaction conditions is mainly determined by the properties of the catalyst (more complex reaction than uncatalyzed reaction). Gibbs (free) energy change, G, tells us whether or not a particular reaction can proceed and how much work it can perform (at a constant temperature and pressure isothermal, isobaric) and tells nothing about rates of chemical reactions.

20 The American mathematical physicist Willard Gibbs

21 Enzymes catalysts of biochemical Increase the rate of chemical reactions by several orders of magnitude acetylcholinesterase molekul s -1 (turnover number) Facilitate reaction course at low temperature, neutral ph and atmospheric pressure They can be easily regulated reactions 1. Proper orientation of substrate 2. Removal of hydration shells 3. Stabilization of transition state 4. Group transfer

22 Progress Curve: Simple Enzyme- Catalyzed Reaction

23 The kinetics of an enzyme-catalyzed reaction S + E ES P + E Michaelis-Menten equation v k K cat E T S S M v K max M S S Michaelis constant K M corresponds to the substrate concentration at which v (rate) is half of the maximum velocity v max (v=v max /2) ; Low K M value means high affinity for its substrate and the other way around. Maximum velocity: v max =k cat [E] T k cat (turnover number): number of molecules of substrate that were transfered by 1 molecule of enzyme per second.

24 Michaelis-Menten Equation [S] low v = (v max /K M ). [S] [S] = K M v = v max /2 [S] high v = v max

25 Hexokinase vs. Glucokinase

26 Double-Reciprocal (Lineweaver-Burk) Plot

27 Values of K m and k cat for some enzymes Activity of enzymes catalytic activity katal [kat] = 1 mol.s -1 ; 1 U = nano katal unit [U] = 1 µmol.min -1

28 Allosteric enzymes Activity of enzymes is modulated by noncovalent bond of specific activator inhibitor Enzyme composed of subunits - hemoglobin cooperative effect hemoglobin monomer enzyme Enzyme formed by subunits

29 Inhibition of enzymes There are numerous substances that modulate activity of enzymes. Of particular importance are enzyme inhibitors. Most enzyme inhibitors act reversibly, i.e. they do not bring about any irreversible change in the enzyme. However, there are also irreversible inhibitors that permanently modify the target enzyme (citation: Color Atlas of Biochemistry). The best method for analyzing of kinetic data that is V max and K M is Double-Reciprocal Plot Lineweaver-Burk equation. The Inhibition of enzymes will be the content of next lecture. Here is only illustration of Competitive Enzyme Inhibition and helping table.

30 Competitive Enzyme Inhibition

31 Competitive Inhibition: Ethanol Treatment of Methanol Poisoning

32 Enzyme Inhibitor Effects