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Introduction into Cell Biology 2 The building blocks of life - Proteins. Intro into cell biology 2. Molecular Organisation of a cell. Fig. 1.7. Building Blocks of Life -> Different Shapes. Proteins Amino Acids are linked by peptide bonds.

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intro into cell biology 2
Intro into cell biology 2

Molecular Organisation of a cell

slide6

Proteins

Amino Acids are linked by peptide bonds

slide11

Proteins are Polypeptides

Direction of a Protein

slide15

Secondary Structure:

1. α – Helix

2. β-Strands -> β-Sheets

3. Loops and Turns

slide17

α-helical coiled coil proteins:

Form superhelix

Found in myosin, tropomyosin (muscle), fibrin (blood clots), keratin (hair)

Examples of α-Helical Proteins:

Hair

slide18

Examples of α-Helical Proteins:

Muscle

α-helical coiled coil proteins:

Form superhelix

Found in myosin, tropomyosin (muscle), fibrin (blood clots), keratin (hair)

slide21

Examples of β-sheet Proteins:

Fatty acid binding protein -> β barrels structure

OmpX: E. coli porin

Antibodies

slide24

Turns and Loops:

Loops in Receptors

Turn

slide26

Quaternary Structure:

Polypeptide chains assemble into multisubunit structures

Cell-surface receptor CD4

Tetramer of hemoglobin

slide27

Protein Folding

Folding is a highly cooperative process (all or none)

Folding by stabilization of Intermediates

slide29

Misfolded protein -> Alzheimer

Protein fibrillation

slide32

Function of Proteins

Specific binding of ligands -> Immunoglobins

slide33

Function of Proteins

Conformational change of lactoferrin upon binding of Fe

Conformational change induced by Calcium

slide34

Function of Proteins

Activation by modification

GFP fluorescent: Rearrangement and oxidation of Ser-Tyr-Gly

slide35

Function of Proteins

Model of enzymatic reaction mechanism

slide36

Proteins Key properties:

  • Proteins are linear polymersbuilt of Amino Acids
  • Proteins contain many functional groups (i.e.. side chain of AA)
  • Proteins interact with proteins and with other biological molecules to form complexes
  • Proteins can bind and/or modify other molecules
  • Proteins can be rigid or can have regions with high flexibility
slide37

Enzyme Kinetics

  • Enzymes DO NOT shift the equilibria but enhance the rates of the reactions (lower the activiation energy!!!)
transition state

X‡

intermediate

H

Reaction coordinate

Transition state
  • Unstable state of maximum energy
  • Not an intermediate
    • Metastable state
  • Intermediates are species that appear in a reaction mechanism but not in the overall balanced equation.

DH‡

H

DH0

Reaction coordinate

slide40

rate =

D[A]

D[B]

rate = -

Dt

Dt

Reaction Kinetics

Thermodynamics – does a reaction take place?

Kinetics – how fast does a reaction proceed?

Reaction rate is the change in the concentration of a reactant or a product with time (M/s).

A B

D[A] = change in concentration of A over

time period Dt

D[B] = change in concentration of B over

time period Dt

Because [A] decreases with time, D[A] is negative.

13.1

slide41

A B

time

rate =

D[A]

D[B]

rate = -

Dt

Dt

13.1

basic problem of enzyme kinetics

Suppose an enzyme were to react with a substrate, giving a product.

Basic problem of enzyme kinetics

S + E

P + E

If we simply applied the law of mass action to this reaction, the rate of reaction would be a linearly increasing function of [S]. As [S] gets very big, so would the reaction rate.

This doesn’t happen. In reality, the reaction rate saturates.

michaelis and menten
Michaelis and Menten

In 1913, Michaelis and Menten proposed the following mechanism for a saturating reaction rate

k1

k2

S + E

ES

P + E

k-1

Complex.

product

slide44

Michaelis-Menten Kinetics

  • When [S] << KM, the reaction increases linearly with [S]; I.e. vo = (Vmax / KM ) [S]
  • Very little [ES] is formed
  • When [S] = KM, vo = Vmax /2 (half maximal velocity); this is a definition of KM: the concentration of substrate which gives ½ of Vmax. This means that low values of KM imply the enzyme achieves maximal catalytic efficiency at low [S].
  • When [S] >> Km, vo = Vmax

Where activity measurements should be performed: 1. [S] very high

2. all enzyme bound in [ES] complex

michaelis menten kinetics
Michaelis-Menten Kinetics

When the enzyme is saturated with substrate, the reaction is progressing at its maximal velocity, Vmax.

Combing the steady-state assumption (d[ES]/dt=0) with the conservation condition ([E]T=[E] + [ES]) vo leads to the Michaelis-Menten Equation of enzyme kinetics:

where Km is

KM= (k-1 + k2)/k1

michaelis menten kinetics46
Michaelis-Menten Kinetics

What is Vmax and KM ?

  • KM gives an idea of the range of [S] at which a reaction will occur. The larger the KM, the WEAKER the binding affinity of enzyme for substrate.
  • Vmax gives an idea of how fast the reaction can occur under ideal circumstances.
michaelis menten kinetics47
Michaelis-Menten Kinetics

Determination of Enzyme kinetics ->

Measure activity (velocity) at different substrate concentrations

Determine activity of an Enzyme ->

Measure at substrate concentration of above 10KM -> no substrate limitation

[E]T=[ES]

michaelis menten kinetics48
Michaelis-Menten Kinetics

How to measure activity of an enzyme using photometrical method ?

Lambert Beer law:

A= c ε l

Where A is the absorbance

c is the concentration (mol/L)

ε is the molar absorption coefficient (L/mol cm)

l is the path length of sample (cm)

Rate = activity = Δc/Δt -> ΔA/Δt = l εΔc/Δt

activity = rate (unit) -> Δc/Δt = (ΔA/Δt)/lε

Definition: 1 Unit of an enzyme will catalyse the reaction of 1 μmol of substrate within 1 min at certian pH and Temperature.

Measure ΔA/Δt (change in absorption/min) for a special enzyme and a high substrate concentration -> from that you can calculate activity of that enzyme at special Temp., solvent, pH, pressure.

michaelis menten kinetics49
Michaelis-Menten Kinetics

How do determine experimentally KM and Vmax ?

(y= d + k x)

Lineweaver-Burk plot

Eadie-Hofstee plot