Transcript Introduction: Protein Aulanni’am Laboratorium Biokimia Jurusan Kimia FMIPA Universitas Brawijaya Aulani " Biokimia" Presentation 11 Introduction: DNA (Genotype) Protein Aulani " Biokimia" Presentation 11

Introduction:
Protein
Aulanni’am
Laboratorium Biokimia Jurusan Kimia
FMIPA
Universitas Brawijaya
Aulani " Biokimia" Presentation 11
Introduction:
DNA
(Genotype)
Protein
Aulani " Biokimia" Presentation 11
Structure…
Aulani " Biokimia" Presentation 11
Structure cont…
Aulani " Biokimia" Presentation 11
Acidic environment
Neutral environment
Alkaline environment
pK2 ~ 9
NH2 H+
R-C-H
COOH
NH2 H+
R-C-H
COOpK1 ~ 2
+1
NH2
R-C-H
COO-
5.5
0
Isoelectric point
Aulani " Biokimia" Presentation 11
-1
Buffer pH
Environment pH vs Protein Charge
10
9
8
7
Isoelectric point,
pI
+
6
5
4
3
0
-
Net
Charge of a Protein
Aulani " Biokimia" Presentation 11
-
pKa of Amino Acids
Amino acids
-COOH -NH2
Gly
Ala
Val
Leu
Ile
Ser
Thr
Met
Phe
Trp
Asn
Gln
Pro
Asp
Glu
His
Cys
Tyr
Lys
Arg
2.34
2.34
2.32
2.36
2.36
2.21
2.63
2.28
1.83
2.38
2.02
2.17
1.99
2.09
2.19
1.82
1.71
2.20
2.18
2.17
G
A
V
L
I
S
T
M
F
W
N
Q
P
D
E
H
C
Y
K
R
-R
pH
two pKa
9.60
pK2
9.69
9.62
9.68
9.68
9.15
pK1
10.4
9.21
9.13
9.39
three pKa
8.80
pK3
9.13
10.6
9.82 3.86
pK2
9.67 4.25
9.17 6.0
?
10.8 8.33
pK1
9.11 10.07
8.95 10.53
9.04 12.48
Aulani " Biokimia" Presentation 11
pI
pK1 + pK2
2
?
pI ?
[OH-]
H
first
HOOC-CH2-C-COOH
Aspartic acid
+1
NH3+
pK1 = 2.1
H
second
HOOC-CH2-C-COO-
Isoelectric point is the average
of the two pKa flanking the
zero net-charged form
2.1 + 3.9
= 3.0
2
Isoelectric point
0
NH3+
pK2 = 3.9
H
-OOC-CH -C-COO2
-1
NH3+ third
pK3 = 9.8
H
-OOC-CH -C-COO2
NH2
-2
-2
pK3
-1
pK2
pK1
Aulani " Biokimia" Presentation 11
0
+1
[OH]
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.
Aulani " Biokimia" Presentation 11
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
Peptide Bonds


Polypeptide = many amino acids
Most proteins have many 100 amino acids
Aulani " Biokimia" Presentation 11
Amino acids are connected head to tail
NH2
1
COOH
NH2
2
COOH
Dehydration
-H2O
O
NH2
1
C N
2
H
Aulani " Biokimia" Presentation 11
COOH
lone pair
electrons
Amino
High pKa Low
N H
H+
H+
N H
H
H
Low pKa High
Carboxylic
C
O H
O
O
C
O
H+
Ampholyte contains both positive and negative groups on its molecule
Aulani " Biokimia" Presentation 11
Levels of Structure…
1 - Primary structure
2 - Secondary structure
3 - Tertiary structure
4 - Quaternary structure
Aulani " Biokimia" Presentation 11
Primary structure…

This is simply the amino acid sequences of
polypeptide chains
Aulani " Biokimia" Presentation 11
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
(C=O) and imido (NH) groups
Aulani " Biokimia" Presentation 11
The -helix

One of the most
closely packed
arrangement of
residues.

Turn: 3.6 residues

Pitch: 5.4 Å/turn
Aulani " Biokimia" Presentation 11
The -sheet

Backbone almost fully extended, loosely
packed arrangement of residues.
Aulani " Biokimia" Presentation 11
Tertiary structure


Three dimensional folding and coiling of polypeptide
into globular 3-D structure
Caused by additional chemical interactions among side
chains
 Disulfide bonds
Aulani " Biokimia" Presentation 11
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
Aulani " Biokimia" Presentation 11
Aulani " Biokimia" Presentation 11
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.
Aulani " Biokimia" Presentation 11
Aulani " Biokimia" Presentation 11
Aulani " Biokimia" Presentation 11
Immune function (antibodies)

Antibodies are proteins that attack and
inactivate bacteria and viruses that cause
infection.
Aulani " Biokimia" Presentation 11
Aulani " Biokimia" Presentation 11
Substrate
Transition state
X
Product
If enzyme just binds substrate
then there will be no further reaction
Enzyme not only recognizes substrate,
but also induces the formation of transition state
Aulani " Biokimia" Presentation 11
The Nature of Enzyme Catalysis
● Enzyme provides a catalytic surface
● This surface stabilizes transition state
● Transformed transition state to product
B
A
A
B
Catalytic surface
Aulani " Biokimia" Presentation 11
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
+
CoE (1)
(4)
(3)
(1) Stabilizes transition
(2)
(2) Expels water
(3) Reactive groups
(4) Coenzyme helps
Aulani " Biokimia" Presentation 11
Enzyme Active Site Is Deeper than Ab Binding
Ag binding site on Ab binds to Ag
complementally, no further reaction
occurs.
Instead, active site on enzyme
also recognizes substrate, but
actually complementally fits the
transition state and stabilized it.
X
Aulani " Biokimia" Presentation 11
Active Site Avoids the Influence of Water
+
-
Preventing the influence of water sustains the formation of stable ionic bonds
Aulani " Biokimia" Presentation 11
Essential of Enzyme Kinetics
Steady State Theory
E
+
S
E
S
E +P
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.
Aulani " Biokimia" Presentation 11
Enzyme Kinetics
Score




0
Student B
Student C
1
2
3 4
Exam Chapters
Enzyme activity
Student A
0 1
2
3 4
Substrate concentration
Increase the substrate concentration,
observe the change of enzyme activity
Aulani " Biokimia" Presentation 11
1
2
3
4
5
6
7
8
S
+
E
↓
P
80
60
40
20
0
0
2
4
6
Substrate (mmole)
Aulani " Biokimia" Presentation 11
8
(in a fixed period of time)
Product
Increase Substrate Concentration
0
An Example for Enzyme Kinetics (Invertase)
1) Use predefined amount of Enzyme
→E
2) Add substrate in various concentrations → S (x 軸)
3) Measure Product in fixed Time (P/t)→ vo (y 軸)
4) (x, y) plot get hyperbolic curve, estimate → Vmax
5) When y = 1/2 Vmax calculate x ([S])
→ Km
Vmax
1
vo
vo
1/2
-1
Km
1
Vmax
1/S" Biokimia" Presentation 11Km
Double reciprocal Aulani
Direct plot
S
A Real Example for Enzyme Kinetics
Data
Substrate Product
Velocity
Double reciprocal
[S] Absorbance v (mmole/min)
0.25
0.21 →
0.42
0.50
0.36 → 0.72
1.0
0.40 → 0.80
2.0
0.46 →
0.92
no
1
2
3
4
1/S
4
2
1
0.5
1/v
2.08
1.56
1.35
1.16
Double reciprocal
Direct plot
(1) The product was measured by spectroscopy at 600 nm for 0.05 per mmole
(2) Reaction time was 10 min
1.0
v
0.5
0
0
1
2
2.0
1.0
1/v
1.0
-3.8
0
-4
Aulani[S]
" Biokimia" Presentation 11
-2
0
2
1/[S]
4
Enzyme Inhibition (Mechanism)
Equation and Description
Cartoon Guide
I
Competitive
I
Non-competitive
Substrate
E
S
S
E
I
Compete for
Inhibitor active site
S
I
I
Uncompetitive
S
E
I
I
Different site
E + S←
→ ES → E + P
+
I
↓↑
EI
E + S←
→ ES → E + P
+
+
I
I
↓↑
↓↑
EI + S →EIS
[I] binds to free [E] only,
and competes with [S];
increasing [S] overcomes
Inhibition by [I].
[I] binds to free [E] or [ES]
complex; Increasing [S] can
not overcome [I] inhibition.
Aulani " Biokimia" Presentation 11
S
I
E + S←
→ ES → E + P
+
I
↓↑
EIS
[I] binds to [ES] complex
only, increasing [S] favors
the inhibition by [I].
Enzyme Inhibition (Plots)
I
Competitive
I
Non-competitive
Direct Plots
Vmax
vo
vo
I
Double Reciprocal
Km Km’
I
[S], mM
Km = Km’
I
Uncompetitive
Vmax
Vmax
Vmax’
Vmax’
[S], mM
I
Km’ Km
[S], mM
Vmax unchanged
Km increased
Vmax decreased
Km unchanged
Both Vmax & Km decreased
1/vo
1/vo
1/vo
Intersect
at Y axis
1/Km
I
I
I
Two parallel
lines
1/ Vmax
1/[S]
Intersect
at X axis
1/Km
1/ Vmax
1/[S]
Aulani " Biokimia" Presentation 11
1/ Vmax
1/Km
1/[S]
1
How to Separate These Objects
Shape
2 3 4 5 6 7 8 9 10 11 12
Size
Density wood stone cotton wood wood cotton stone wood stone cotton stone cotton
1
2 3
Shape
6
Size
4 5 6
4
7 8
Density
5
cotton
8
7
9
10 11
4
8
wood
stone
5
12
Different sedimentation
Sieving different sizes
Aulani " Biokimia" Presentation 11
Different rolling speed
Basic Principles of Protein Purification
Organelle
Cell
Homogenization
Macromolecule
Small molecule
Amino acid, Sugar,
Nucleotides, etc
Nucleic
acid
Protein
Ammonium sulfate
Size
Gel filtration,
SDS-PAGE,
Ultrafiltration
Charge
Carbohydrate
(Lipid)
Cell
Debris
fractionation
Polarity
Affinity
Ion exchange,
Reverse phase
Affinity
Chromatofocusing, chromatography, chromatography,
Disc-PAGE,
Salting-out
Hydroxyapatite
Isoelectric focusing
Aulani " Biokimia" Presentation 11
Thank you
Aulani " Biokimia" Presentation 11