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
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.

4. 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 

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 
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 

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

11. Substrate Binding / Active Site
Lysozyme

12. Enzymatic Reaction
Substrate ‘A’
Substrate ‘B’
Enzyme

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

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

15. Substrates Affect Enzymes on Binding
Background
‘B’ Has Become ‘D’
‘A’ Has Become ‘C’
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. 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

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

19. Substrate Binding

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

21. 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)

22. 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)

23. 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

24. 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

25. 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

26. 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

27. Multiple Substrate Reactions - Double Displacement
Two things are happening

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

29. 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