Cell Division Mitosis

1
Introduction
Protein
Aulanniam Laboratorium Biokimia Jurusan
Kimia FMIPA Universitas Brawijaya
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Introduction

3
Structure
4
Structure cont
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Acidic environment
Neutral environment
Alkaline environment
NH2 H
COOH
5.5
0
1
-1
Isoelectric point
6
Environment pH vs Protein Charge
Buffer pH
Isoelectric point, pI

-
0
-
Net Charge of a Protein
7
pKa of Amino Acids
Amino acids -COOH -NH2 -R Gly G 2.34 9.60 Ala A
2.34 9.69 Val V 2.32 9.62 Leu L 2.36 9.68 Ile I
2.36 9.68 Ser S 2.21 9.15 Thr T 2.63 10.4 Met
M 2.28 9.21 Phe F 1.83 9.13 Trp W 2.38 9.39 Asn
N 2.02 8.80 Gln Q 2.17 9.13 Pro P 1.99 10.6 As
p D 2.09 9.82 3.86 Glu E 2.19 9.67 4.25 His H 1.82
9.17 6.0 Cys C 1.71 10.8 8.33 Tyr Y 2.20 9.11 10.
07 Lys K 2.18 8.95 10.53 Arg R 2.17 9.04 12.48
two pKa
pK2
pI
pK1
three pKa
pK3
?
pK2
?
pI ?
pK1
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Aspartic acid
first
1
Isoelectric point is the average of the two pKa
flanking the zero net-charged form
pK1 2.1
second
0
3.0
Isoelectric point
pK2 3.9
-1
third
pK3 9.8
-2
OH
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Protein?

• Protein are linear heteropolymers one or more
  polypeptide chains
• Building blocks 20(?) amino acid residues.
• Range from a few 10s-1000s
• Three-dimensional shapes (fold) adopted vary
  enormously.

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Primary structure of a protein

• It is the sequence of amino acids that makes each
  protein different from the next
• Dipeptide 2 amino acids
• Tripeptide 3 amino acids
• Polypeptide many amino acids
• Most proteins have many 100 amino acids

Peptide Bonds
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Amino acids are connected head to tail
Dehydration -H2O
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lone pair electrons
pKa
H
pKa
Ampholyte contains both positive and negative
groups on its molecule
13
Levels of Structure

• 1 - Primary structure
• 2 - Secondary structure
• 3 - Tertiary structure
• 4 - Quaternary structure

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Primary structure

• This is simply the amino acid sequences of
  polypeptide chains

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Secondary structure

• Local organization of protein backbone ?-helix,
  ?-strand (which assemble into ?-sheet), turn and
  interconnecting loop.

• Alignment of polypeptides as a right-hand alpha
  helix
• Stabilized by hydrogen bonds between carboxyl
  (CO) and imido (NH) groups

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The ?-helix

• One of the most closely packed arrangement of
  residues.
• Turn 3.6 residues
• Pitch 5.4 Å/turn

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The ?-sheet

• Backbone almost fully extended, loosely packed
  arrangement of residues.

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Tertiary structure

• Three dimensional folding and coiling of
  polypeptide into globular 3-D structure
• Caused by additional chemical interactions among
  side chains
• Disulfide bonds

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Quaternary structure

• Assembly of homo or heteromeric protein chains.
• Usually the functional unit of a protein,
  especially for enzymes

• Interactive folding of several polypeptide chains
  together to form a single functional protein
• Functional proteins also might incorporate
  minerals or other nonprotein components

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Enzymes

• Proteins that catalyze (speed up) chemical
  reactions without being used up or destroyed in
  the process.
• Anabolic (putting things together) and catabolic
  (breaking things down) functions.

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Immune function (antibodies)

• Antibodies are proteins that attack and
  inactivate bacteria and viruses that cause
  infection.

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Substrate
Transition state
Product
If enzyme just binds substrate then there will
be no further reaction
X
Enzyme not only recognizes substrate, but also
induces the formation of transition state
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The Nature of Enzyme Catalysis
? Enzyme provides a catalytic surface
? This surface stabilizes transition state
? Transformed transition state to product
B
A
Catalytic surface
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Active Site Is a Deep Buried Pocket
Why energy required to reach transition state is
lower in the active site?
It is a magic pocket

(1) Stabilizes transition
CoE
(2) Expels water
(2)
(1)
(3) Reactive groups
(4)
-
(4) Coenzyme helps
(3)
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Enzyme Active Site Is Deeper than Ab Binding
Instead, active site on enzyme also recognizes
substrate, but actually complementally fits the
transition state and stabilized it.
Ag binding site on Ab binds to Ag complementally,
no further reaction occurs.
X
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Active Site Avoids the Influence of Water

-
Preventing the influence of water sustains the
formation of stable ionic bonds
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Essential of Enzyme Kinetics
Steady State Theory
E
E


P
S
In steady state, the production and consumption
of the transition state proceed at the same rate.
So the concentration of transition state keeps a
constant.
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Enzyme Kinetics
Student A
Enzyme activity
Score
Student B
Student C
0 1 2 3 4
0 1 2 3 4
Substrate concentration
Exam Chapters
Increase the substrate concentration, observe the
change of enzyme activity
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Increase Substrate Concentration
S E ? P
(in a fixed period of time)
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An Example for Enzyme Kinetics (Invertase)
1) Use predefined amount of Enzyme ? E
2) Add substrate in various concentrations ? S
(x ?)
3) Measure Product in fixed Time (P/t) ? vo (y ?)
4) (x, y) plot get hyperbolic curve, estimate ?
Vmax
5) When y 1/2 Vmax calculate x (S) ? Km
Vmax
1/2
Km
Double reciprocal
Direct plot
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A Real Example for Enzyme Kinetics
Data
v (mmole/min)
S
Absorbance
1/S
1/v
no
0.25 0.50 1.0 2.0
0.42 0.72 0.80 0.92
0.21 0.36 0.40 0.46
4 2 1 0.5
2.08 1.56 1.35 1.16
1 2 3 4
? ? ? ?
(1) The product was measured by spectroscopy at
600 nm for 0.05 per mmole (2) Reaction time was
10 min
Direct plot
Double reciprocal
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Enzyme Inhibition (Mechanism)
Competitive
Non-competitive
Uncompetitive
E
Substrate
E
X
Cartoon Guide
Compete for active site
Inhibitor
Different site
Equation and Description
I binds to free E only, and competes with
S increasing S overcomes Inhibition by I.
I binds to ES complex only, increasing S
favors the inhibition by I.
I binds to free E or ES complex
Increasing S can not overcome I inhibition.
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Enzyme Inhibition (Plots)
Vmax
vo
I
I
Km
Km
S, mM
Km
Vmax unchanged Km increased
Vmax decreased Km unchanged
Both Vmax Km decreased
I
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How to Separate These Objects
Shape Size Density
wood stone cotton wood wood cotton stone wood
stone cotton stone cotton
Shape
4
6
Size
8
5
Density
7
Different rolling speed
Different sedimentation
Sieving different sizes
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Basic Principles of Protein Purification
Organelle
Homogenization
Small molecule
Cell Debris
Amino acid, Sugar, Nucleotides, etc
Protein
Nucleic acid
Carbohydrate
(Lipid)
Ammonium sulfate fractionation
Size
Charge
Polarity
Affinity
Ion exchange, Chromatofocusing, Disc-PAGE, Isoelec
tric focusing
Reverse phase chromatography, Salting-out
Gel filtration, SDS-PAGE, Ultrafiltration
Affinity chromatography, Hydroxyapatite
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Thank you