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