Enzymes and Enzyme Kinetics I Dr.Nabil Bashir

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

Enzymes and Enzyme Kinetics I: Outlines…….. Free Energy G determines the direction a reaction proceeds 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. G0' G0' is related to  G. It is the same as  G under standard conditions Thus, the sign of  G0' determines the direction of a reaction ONLY under standard conditions.  G determines the direction of a reaction under any conditions, including standard conditions.

Enzymes and Enzyme Kinetics I: Outlines…….. Calculations G =  G0' + RT ln[Products]/[Reactants], w R is the gas constant and T is the temperature in Kelvin At equilibrium,  G = 0, so  G0' = -RT ln[Products]/[Reactants] = -RTln K'eq Thus,  G0' is related to the equilibrium constant for a reaction Relation between  G0' and K'eq at 25°C 

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 

Enzymes and Enzyme Kinetics I: Outlines…….. Michaelis-Menten Model Initial velocity determination  KM determination  LineWeaver-Burk plot  KM 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 

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 Triose phosphate isomerase by bromoacetal phosphate  Glycopeptide transpeptidase by penicillin 

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

Enzymatic Reactions

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

Substrate Binding / Active Site Lysozyme

Enzymatic Reaction Substrate ‘A’ Substrate ‘B’ Enzyme

Enzymatic Reaction Substrate ‘A’ Substrate ‘B’ Enzyme ES Complex

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

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

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

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

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

Substrate Binding

Concerted Model of Catalysis Enzymes Catalysis Considerations - Models Concerted Model of Catalysis

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

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

Enzymes lower activation energy Enzymes catalyze reversible reactions 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

A <=> B At equilibrium, [A]T0 = [B]T0 [A]T0 = [A]T+5 Similarly, 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

Types of Reactions Substrate Binding 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

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

Multiple Substrate Reactions - Double Displacement Two things are happening

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

E+S <=> ES <=> ES* <=> EP <=> E+P 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