enzymes and enzyme kinetics i - كلية الطب · 2020-01-22 · enzymes and enzyme kinetics i:...
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Enzymes and Enzyme Kinetics I
Dr.Nabil Bashir
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
o If G <0, the reaction proceeds forward as written
o If G >0, the reaction proceeds backward as written
o If G = 0, the reaction is at equilibrium
o 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'
o G0' is related to G. It is the same as G under standard conditions
o 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
o G = G0' + RT ln[Products]/[Reactants], w R is the gas constant and T is
the temperature in Kelvin
o At equilibrium, G = 0, so G0' = -RT ln[Products]/[Reactants] = -RTln
K'eq
o Thus, G0' is related to the equilibrium constant for a reaction
o 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
o Triose phosphate isomerase by bromoacetal phosphate
o Glycopeptide transpeptidase by penicillin
Enzymes and Enzyme Kinetics I: Outlines……..
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
Enzymes and Enzyme Kinetics I: specific aims
Enzymatic Reactions
Enzymatically Catalyzed Reactions • Background
• Enzyme
• Substrate(s)
• Active Site
Substrates Bound at Active Site of the
Methylene Tetrahydrofolate Reductase Enzyme
Active Site
Substrates
Substrate Binding / Active Site
Substrate
Substrate
Binding
Site
Active Site
Lysozyme
Enzyme
Substrate ‘A’
Substrate ‘B’
Enzymatic Reaction
Enzyme
Substrate ‘A’
Substrate ‘B’
ES Complex
Enzymatic Reaction
Enzymatic Reaction
Enzyme Changes Result
in Reaction
Between
Substrates A and B
ES* Complex
Substrates Affect Enzymes on Binding • Background
EP Complex
Part of ‘A’
Has Moved to ‘B’
‘A’ Has Become ‘C’ ‘B’ Has Become ‘D’
Substrates Affect Enzymes on Binding • Background
E + P
Product C
Released
Product D Released
Enzyme Freed to Bind
More Substrates
Steps In Catalysis • Background
E + S Free Enzyme
& Substrates
<=
>
ES Substrate Binding
<=
>
ES* Reaction
<=
>
EP Product Formation
<=
>
E + P Release of Products
Reversible
Enzymes • Mechanistics
• Binding Specificity
• Flexibility
• Electronic Environment
• Coenzymes
A Serine Protease
Substrate Binding
Enzymes • Catalysis Considerations - Models
Concerted Model of Catalysis
Enzyme Action
Free Energy of
Substrate(s)
Activation Energy
Necessary for
Uncatalyzed Reaction
Free Energy of
Product(s)
Overall
Free
Energy
Change
Reaction
Goes
Forward
Reaction
Reverses
Free Energy of
Substrate(s)
Activation Energy
Necessary for Uncatalyzed Reaction
Reaction
Goes
Forward
Reaction
Reverses
Free Energy of
Product(s)
Activation Energy
Necessary for Catalyzed
Reaction
No Overall
Free
Energy
Change
Enzyme Action
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
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
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
Multiple Substrate Binding
Ordered
Lactate
Dehydrogenase
NADH + Pyruvate
Lactate + NAD+ Must
bind
first
Random
Creatine + ATP
Creatine phosphate + ADP
Creatine
Kinase
No order
to binding
Multiple Substrate Reactions - Double Displacement
Two things are happening
Enzymes • Ping-Pong Catalysis
Enzyme Flips Between Two States
Enzyme Flips Between Two States
Enzymes • Kinetic Considerations
E+S <=> ES <=> ES* <=> EP <=> E+P
Rate of Formation of Product of Primary Interest
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