Transcript 2 HI

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Note: Graphic 24 has been modified from the original posting:
(“Most spherical” was replaced by “least spherical”).
Columbia Biological Society
First meeting:
Tuesday Sep 25
at 9pm
in 702 Hamilton.
Some prosthetic groups
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Particular small molecules so tightly bound that they are always
found associated with the protein
Tetrahydrofolic acid
~ vitamin B9
Pyridoxal phosphate
~ vitamin B6
Riboflavin
~ vitamin B2
Heme
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Membrane proteins
Could be size selective
Could be size and charge selective
Anion: an ion that would migrate to the anode in an electric field
Small molecules bind with great specificity to pockets on protein surfaces
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Too far
Estrogen receptor binding estrogen, a steroid hormone
detail
estrogen
estrogen
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Protein separation methods
Ultracentrifugation
Mixture of proteins
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Causing sedimentation:
centrifugal force = m(omega)2r
m = mass
omega = angular velocity
r = distance from the center of rotation
Opposing sedimentation = friction = foV.
Constant velocity is soon reached:
centrifugal force = frictional force
So:
m(omega)2r = foV
fo = frictional coefficient (depends on shape)
And: V = m(omega)2r/fo,
Or: V = [(omega)2r] x [m / fo]
Note: formulas wil be provided on exams, as will formulae
V proportional to mass (MW)
V inversely proportional to fo (shape)
V inversely proportional to non-sphericity
(Spherical shape moves fastest)
Ultracentrifuge
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+
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Glass
plates
+
+
Large, +++
high positive charge
+
Large, +
low positive charge
++
+
+
+
++
+
Small, +++
High positive charge
+ ++
+
+
Small, +
Low positive charge
+ +
+
+ +
+
+ +
+
+
Molecules shown after several
hours of electrophoresis
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Glass
plates
+
+
++
+
+ +
+
+
+
Winner:
Small, +++
High positive charge
+
++
+
+ +
+
+ ++
+
+
+ +
+
Loser:
Large, +
low positive charge
Intermediate:
Large, +++
high positive charge
Intermediate:
+
Small,
+
Low positive charge
Molecules shown after several
hours of electrophoresis
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Electrode connection
Power supply
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Electrodes
Tracking dyes
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SDS PAGE = SDS polyacrylamide gel
electrophoresis
• sodium dodecyl sulfate, SDS (or SLS): CH3-(CH2)11- SO4•
CH3-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-SO4-
SDS
All the polypeptides are denatured and behave as random coils
All the polypeptides have the same charge per unit length
All are subject to the same electromotive force in the electric field
Separation based on the sieving effect of the polyacrylamide gel
Separation is by molecular weight only
SDS does not break covalent bonds (i.e., disulfides)
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Summary of SDS PAGE
Separates on MW only, no shape no charge
High resolution.
Can measure the MW of a protein (subunit MW) by comparig mobiltiy to that of standards.
Must first reduce any disulfides to get true subunit MW (e.g., with mercaptoethanol).
Molecular sieve chromatography
(=gel filtration, =Sephadex chromatography)
Sephadex bead
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Molecular sieve chromatography
Sephadex bead
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Molecular sieve chromatography
Sephadex bead
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Molecular sieve chromatography
Sephadex bead
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Molecular sieve chromatography
Sephadex bead
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Plain column of Sephadex
Fancy column of Sephadex
Handout 4-3: protein separations
Handout
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Winners:
Largest and
most spherical
Lowest MW
Largest and
least spherical
Similar to handout,
but Winners &
Native PAGE added
Most charged
and smallest
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Enzymes =
protein catalysts
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Flow of glucose in E. coli
Macromolecules
Polysaccharides
Lipids
Nucleic Acids
Proteins
yn
th
e
tic
pa
t
hw
ay
monomers
bi
os
intermediates
glucose
Each arrow = an ENZYME
Each arrow = a specific chemical reaction
Chemical reaction between 2 reactants
H2 + I2
2 HI
H2 + I2
2 HI + energy
“Spontaneous” reaction:
Energy released
Goes to the right
H-I is more stable than H-H or I-I here
That’s why it “goes’ to the right,
i.e., it will end up with more products than reactants
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say, 100
kcal/mole
say, 103
kcal/mole
H2 + I2
2 HI
{
Change in Energy (Free Energy)
2H + 2I
Atom pulled completely apart
(thought experiment)
-3 kcal/mole
Reaction goes spontaneously to the right
Energy change is negative: spontaneously to the right = exergonic: energy-releasing
Energy change is positive: spontaneously to the left = endergonic: energy-requiring
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H2 + I2
2 HI
H2 + I2
2 HI
H2 + I2
2 HI
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But: it is not necessary to break molecule down
to its atoms in order to rearrange them
say, 100
kcal/mole
say, 103
kcal/mole
H2 + I2
2 HI
{
Change in Energy (Free Energy)
2H + 2I
-3 kcal/mole
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I
I
+
H H
I
I
+
H H
I
I
H H
I
I
H
H
Transition state
(TS)
(H2 + I2)
I
H
+
I
H
(2 HI)
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Change in Energy
2H + 2I
~100 kcal/mole
H-H
| |
I-I
(TS)
Say,
~20 kcal/mole
2 HI
{
H2 + I2
-3 kcal/mole
Activation
Energy
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Allows it to happen
Energy needed
to bring molecules
together to form
a TS complex
determines speed =
VELOCITY =
rate of a reaction
H2 + I2
2 HI
{
Change in Energy (new scale)
HHII
(TS)
3 kcal/mole
Net energy change:
Which way it
will end up
DIRECTION
of the reaction, independent of the rate
Activation
energy
Biosynthesis of a fatty acid
3 glucose
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18-carbon fatty acid
Free energy change: ~ 300 kcal per mole of glucose is REQUIRED
3 glucose
18-carbon fatty acid
So getting a reaction to go in the direction you want is a problem
(to be discussed next time)
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Concerns about the cell’s chemical reactions
• Direction
– We need it to go in the direction we want
• Speed
– We need it to go fast enough to have the
cell double in one generation
– Catalysts deal with this second problem, which we will now
consider
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The velocity problem is solved by catalysts
The catalyzed reaction
The catalyst takes part in the reaction,
but it itself emerges unchanged
Change in Energy
HHII
(TS)
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Activation
energy
without
catalyst
TS
complex
with
catalyst
H2 + I2
2 HI
Activation
energy
WITH the
catalyst
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Reactants in an enzyme-catalyzed reaction = “substrates”
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Reactants (substrates)
Not a substrate
Active site
or
substrate binding site
(not exactly synonymous,
could be part of the active site)
Unlike inorganic catalysts,
Enzymes are specific
succinic dehydrogenase
HOOC-HC=CH-COOH <--------------------------------> HOOC-CH2-CH2-COOH
+2H
fumaric acid
succinic acid
NOT a substrate for the enzyme:
1-hydroxy-butenoate: HO-CH=CH-COOH
(simple OH instead of one of the carboxyls)
Maleic acid
Platinum will work with all of these, indiscriminantly
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Enzymes work as catalysts for two reasons:
1. They bind the substrates putting them in close proximity.
2. They participate in the reaction, weakening the covalent bonds
of a substrate by its interaction with the enzyme’s amino acid side
groups (e.g., stretching).
Chemical kinetics
Substrate  Product
SP
Velocity = V = ΔP/ Δ t
(reactants in enzyme catalyzed reactions are called substrates)
So V also = -ΔS/ Δt (disappearance)
From the laws of mass action:
ΔP/ Δt = - ΔS/ Δt = k1[S] – k2[P]
back reaction
For the INITIAL reaction, [P] is small and can be neglected:
ΔP/ Δt = - ΔS/ Δt = k1[S]
So the INITIAL velocity Vo = k1[S]
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Vo = the slope in each case
Effect of different initial
substrate concentrations
0.6
[S4]
[S3]
P
0.4
[S2]
0.2
[S1]
0.0
t
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Considering Vo as a function of [S]
(which wil be our usual useful consideration)