Title: Introduction into Cell Biology 2 The building blocks of life - Proteins
1Introduction into Cell Biology 2The building
blocks of life - Proteins
2Intro into cell biology 2
Molecular Organisation of a cell
3Fig. 1.7
4Building Blocks of Life -gt Different Shapes
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6Proteins Amino Acids are linked by peptide bonds
720 Natural Occurring Amino Acids are divided
into groups according to their side chains
8The Aromatic Amino Acids
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10Cys can cross-link between 2 polypeptide chains
-gt Disulfide bridge
11Proteins are Polypeptides
Direction of a Protein
123D Structure of Proteins
13Interactions between side chains and backbone -gt
Fold of a protein (3D structure)
14Noncovalent interactions within and between
biological molecules
15Secondary Structure
1. a Helix 2. ß-Strands -gt ß-Sheets 3. Loops
and Turns
16a-Helix
17Examples of a-Helical Proteins
Hair
18Examples of a-Helical Proteins
Muscle
a-helical coiled coil proteins Form
superhelix Found in myosin, tropomyosin (muscle),
fibrin (blood clots), keratin (hair)
19Examples of a-Helical Proteins
20ß-Strands -gt ß-sheets
21Examples of ß-sheet Proteins
Fatty acid binding protein -gt ß barrels structure
Antibodies
OmpX E. coli porin
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24Turns and Loops
Loops in Receptors
Turn
25Tertiary Structure 3D structure of a polypeptide
chain
26Quaternary Structure Polypeptide chains
assemble into multisubunit structures
Cell-surface receptor CD4
Tetramer of hemoglobin
27Protein Folding
Folding is a highly cooperative process (all or
none)
Folding by stabilization of Intermediates
28Protein Folding by Chaperons
29Misfolded protein -gt Alzheimer
Protein fibrillation
30Function of Proteins
31Molecular Machines Transcription Initiation
Complex
32Function of Proteins
Specific binding of ligands -gt Immunoglobins
33Function of Proteins
Conformational change of lactoferrin upon binding
of Fe
Conformational change induced by Calcium
34Function of Proteins
Activation by modification
GFP fluorescent Rearrangement and oxidation of
Ser-Tyr-Gly
35Function of Proteins
Model of enzymatic reaction mechanism
36Proteins Key properties
- Proteins are linear polymers built 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
37Enzyme Kinetics
- Enzymes DO NOT shift the equilibria but enhance
the rates of the reactions (lower the activiation
energy!!!)
38Transition 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
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40Reaction 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).
DA change in concentration of A over
time period Dt
DB change in concentration of B over
time period Dt
Because A decreases with time, DA is
negative.
13.1
4113.1
42Basic problem of enzyme kinetics
Suppose an enzyme were to react with a substrate,
giving a product.
43Michaelis and Menten
In 1913, Michaelis and Menten proposed the
following mechanism for a saturating reaction rate
Complex.
product
44Michaelis-Menten Kinetics
- When S ltlt 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 gtgt Km, vo Vmax
Where activity measurements should be performed
1. S very high
2. all enzyme bound in ES complex
45Michaelis-Menten Kinetics
When the enzyme is saturated with substrate, the
reaction is progressing at its maximal velocity,
Vmax. Combing the steady-state assumption
(dES/dt0) with the conservation condition
(ETE ES) vo leads to the
Michaelis-Menten Equation of enzyme kinetics
where Km is
KM (k-1
k2)/k1
46Michaelis-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.
47Michaelis-Menten Kinetics
Determination of Enzyme kinetics -gt Measure
activity (velocity) at different substrate
concentrations Determine activity of an Enzyme
-gt Measure at substrate concentration of above
10KM -gt no substrate limitation ETES
48Michaelis-Menten Kinetics
How to measure activity of an enzyme using
photometrical method ? Lambert Beer law
A c e
l Where A is the absorbance c is the
concentration (mol/L) e is the molar
absorption coefficient (L/mol cm) l
is the path length of sample (cm)
Rate activity ?c/?t -gt ?A/?t l e
?c/?t activity rate (unit)
-gt ?c/?t (?A/?t)/le Definition 1 Unit of an
enzyme will catalyse the reaction of 1 µmol of
substrate within 1 min at certian pH and
Temperature. Measure ?A/?t (change in
absorption/min) for a special enzyme and a high
substrate concentration -gt from that you can
calculate activity of that enzyme at special
Temp., solvent, pH, pressure.
49Michaelis-Menten Kinetics
How do determine experimentally KM and Vmax ?
Lineweaver-Burk plot
Eadie-Hofstee plot