Enzymes and Enzyme Kinetics I. Dr.Nabil Bashir

Transcription

1 Enzymes and Enzyme Kinetics I Dr.Nabil Bashir

2 Enzymes and Enzyme Kinetics I: Outlines Enzymes - Basic Concepts and Kinetics Enzymes as Catalysts Enzyme rate enhancement / Enzyme specificity Enzyme cofactors

3 Enzymes and Enzyme Kinetics I: Outlines.. Free Energy G determines the direction a reaction proceeds o o o o G 0 ' o o If G <0, the reaction proceeds forward as written If G >0, the reaction proceeds backward as written If G = 0, the reaction is at equilibrium Note that equilbrium DOES NOT mean equal concentrations of reactants and products. Rather, equilbrium means that the concentration of reactants and products does not change over time. G 0 ' is related to G. It is the same as G under standard conditions Thus, the sign of G 0 ' determines the direction of a reaction ONLY under standard conditions. G determines the direction of a reaction under any conditions, including standard conditions.

4 Enzymes and Enzyme Kinetics I: Outlines.. Calculations o o o G = G 0 ' + RT ln[products]/[reactants], w R is the gas constant and T is the temperature in Kelvin At equilibrium, G = 0, so G 0 ' = -RT ln[products]/[reactants] = -RTln K'eq Thus, G 0 ' is related to the equilibrium constant for a reaction o Relation between G 0 ' and K'eq at 25 C

5 Enzymes and Enzyme Kinetics I: Outlines.. Mechanisms Enzymes speed reactions Enzymes act by decreasing activation energy Reaction velocity versus substrate concentration Residues at active site / Hydrogen bonds with substrate Fischer lock&key model of catalysis / Koshland induced fit model

6 Enzymes and Enzyme Kinetics I: Outlines.. Michaelis-Menten Model Initial velocity determination K M determination LineWeaver-Burk plot K M values of some enzymes / Turnover numbers Substrate preferences of chymotrypsin Diffusion-controlled enzymes Multiple Substrate Reactions Sequential displacement Ordered example / Schematic Random example / Schematic Double displacement Allosteric enzyme kinetics

7 Enzymes and Enzyme Kinetics I: Outlines.. Enzyme Inhibition Uninhibited vs. Competitive vs. Uncompetitive vs. Non-Competitive Methotrexate and Tetrahydrofolate Kinetics of a competitive inhibitor (V vs. [S]) and (Lineweaver-Burk) Kinetics of a non-competitive inhibitor (V vs. [S]) and (Lineweaver-Burk) Serine modification by DIPF Suicide inhibition o o Triose phosphate isomerase by bromoacetal phosphate Glycopeptide transpeptidase by penicillin

8 Enzymes and Enzyme Kinetics I: specific aims You must be able to: 1. Compare uncatalyzed reactions with enzymatic catalyzed reactions and appreciate the rate of enhancement done by enzymes 2. Define substrate, substrate binding site and active site 3. State and understand the steps in enzyme catalytic reactions: E+S <=> ES <=> ES* <=> EP <=> E+P 4. Define the terms Binding Specificity, Flexibility, Electronic Environment, and Coenzymes and recognize their importance in enzyme action 5. Differentiate between substrate binding models; fisher and koshland, 6.Correlate tension to koshland mechanism 7. Understand the energy profile of catalyzed reactions 8. Know that enzymes lower the activation energy without affecting the energy of the whole reaction and thus not affecting K equiliprium 9.State the types of enzymatic reactions and know what happened in each case 10. Define and understand the ordered, random, and ping-pong reactions of the multiple substrate multiple products 11. Understand that n kinetics means measuring rate of product formation, specifically Kcat

9 Enzymatic Reactions

10 Enzymatically Catalyzed Reactions Background Substrates Enzyme Substrate(s) Active Site Active Site Substrates Bound at Active Site of the Methylene Tetrahydrofolate Reductase Enzyme

11 Substrate Binding / Active Site Substrate Active Site Lysozyme Substrate Binding Site

12 Enzymatic Reaction Substrate A Substrate B Enzyme

13 Enzymatic Reaction Substrate A Substrate B Enzyme ES Complex

14 Enzymatic Reaction Enzyme Changes Result in Reaction Between Substrates A and B ES* Complex

15 Substrates Affect Enzymes on Binding Background A Has Become C B Has Become D Part of A Has Moved to B EP Complex

16 Substrates Affect Enzymes on Binding Background Product C Released Product D Released Enzyme Freed to Bind More Substrates E + P

17 <=> <=> > =< > =< Steps In Catalysis Background E + S Free Enzyme & Substrates ES Substrate Binding Reversible ES* Reaction EP Product Formation E + P Release of Products

18 Enzymes Mechanistics Binding Specificity Flexibility Electronic Environment Coenzymes A Serine Protease

19 Substrate Binding

20 Enzymes Catalysis Considerations - Models Concerted Model of Catalysis

21 Enzyme Action Free Energy of Substrate(s) Activation Energy Necessary for Uncatalyzed Reaction Reaction Reverses Reaction Goes Forward Overall Free Energy Change Free Energy of Product(s)

22 Enzyme Action Activation Energy Necessary for Uncatalyzed Reaction Free Energy of Substrate(s) Activation Energy Necessary for Catalyzed Reaction Reaction Reverses Reaction Goes Forward No Overall Free Energy Change Free Energy of Product(s)

23 Enzymatic Reactions Enzymes lower activation energy Enzymes catalyze reversible reactions Enzymes do not change overall energy Enzymes do not change equilibrium concentrations Enzymes speed achieving equilibrium

24 Enzymatic Reactions A <=> B At equilibrium, [A]T0 = [A]T+5 Similarly, [B]T0 = [B]T+5 At any amount of time X after equilibrium has been reached, [A]T0 = [A]T+5 = [A]TX and [B]T0 = [B] T+5 = [B]TX However, unless ΔG = 0, it is wrong to say [A]T0 = [B]T0

25 Substrate Binding Types of Reactions Single Substrate - Single Product : A B Single Substrate - Multiple Products : A B + C Multiple Substrates - Single Products : A + B C Multiple Substrates - Multiple Products : A + B C + D Ordered Random Ping-Pong

26 Multiple Substrate Binding Ordered Random Must bind first NADH + Pyruvate Lactate + NAD + Lactate Dehydrogenase Creatine + ATP Creatine phosphate + ADP Creatine Kinase No order to binding

27 Multiple Substrate Reactions - Double Displacement Two things are happening

28 Enzymes Ping-Pong Catalysis Enzyme Flips Between Two States Enzyme Flips Between Two States

29 Enzymes Kinetic Considerations Rate of Formation of Product of Primary Interest E+S <=> ES <=> ES* <=> EP <=> E+P If We Assume in the Simple Case that ES Proceeds Directly to E+P, where kf, kr, and kcat refer to the rate constants for formation of ES, reversible breakdown of ES, and conversion of ES to E+P, respectively