Physical Properties - Winthrop University

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Protein Structure: Myoglobin as an Example
• John Kendrew solved the structure of myoglobin in 1959
– No Computers!
• Myoglobin is a globular protein consisting of 153 amino
acids and a prosthetic group: A Heme
• Myoglobin has no -sheets and is 100% -helical with
respect to secondary structure
(we won’t count turns)
– There are 8 helices, labelled A through H
• Polar amino acids are on the surface and hydrophobic
amino acids are in the core of the protein
– This is a standard arrangement in proteins
• Two histidines help lock the heme group into position
• Hydrophobic interactions b/w the heme group and
hydrophobic amino acids in the core complete the binding
of the heme
The Heme Group
• Heme consists of a metal
ion, Fe (II), and a porphyrin
ring
– The ring is a planar structure
• Fe (II) can accommodate 6
coordinate bonds, forming
an octahedral arrangement
– The porphyrin nitrogens
provide 4 of these
• An imidazole nitrogen of a
histidine in helix F provides
a 5th bond
• Oxygen binds to Fe (II) to
complete the arrangement
Hydrophobic
Interactions help
anchor the porphyrin
ring
His 97 and Arg 45
help anchor the ring
His 93 Interacts with
the Fe (II)
The histidine above the porphyrin ring (on the same side of the ring as where
oxygen binds)
This off-centered binding forces any other molecule that would bind to the Fe
(II) to bind less optimally
•Carbon monoxide is a good example
•It also allows oxygen to dissociate formt he iron
•If the binding was too strong, the Fe (II) - Oxygen bond wouldn’t break
Quick Thoughts on Protein Folding
• There are literally millions of possible ways a simple protein
can fold, but only one conformation that works
• Hydrophobic interactions help drive protein folding
– Not so much the hydrophobic groups attracting each other (That only involves
London Forces, right?)
– The dipole-dipole interactions between water molecules in solution
are much stronger and push the hydrophobic side chains aside
• The entropy of the universe must increase in a spontaneous
process, and protein folding is a spontaneous process
– When water molecules surround a nonpolar compound, they are
restricted in the number of hydrogen bonds then can form which
represents a lower entropy
– By having the hydrophobic residues sequestered in the core of the
folded protein, the water molecules are free to form up to 4 hydrogen
bonds each.
• This freedom represents greater entropy, thus helping drive
folding of the protein
Protein Folding: Myoglobin
QuickTime™ and a
YUV420 codec decompressor
are needed to see this picture.
Note Placement of hydrophobic residues (green)
Where are the side chains pointing?
QuickTime™ and a
YUV420 codec decompressor
are needed to see this picture.
Note Placement of polar residues (blue)
Where are the side chains pointing?
Chapter 6: Enzymes as Catalysts
• Proteins perform many functions in the cell:
– Structural roles, Signalling, DNA binding, Energy
Transduction, Metabolism and many, many more
• Perhaps the most important role proteins play
is to serve as catalysts
– Enzymes are protein catalysts
– Enzymes increase the reaction rate by up to 1020
times
• Non-enzymatic catalysts typically only increase the rate
100 to 10000 fold
Kinetics versus Thermodynamics
Standard Free Energy of Reaction
G°= G°Products - G°Reactants
• The reaction rate depends of the
Activation Energy (EA):
EA= G°Transition State - G°Reactants
• An enzyme lowers the activation
energy
– Helps substrate move to
transition state
Enzyme Catalyzed Reactions
• An enzyme cannot make a nonspontaneous
reaction occur
• Let’s look at the reaction of glucose and
oxygen:
C6H12O6 + 6O2 --> 6CO2 + 6H2O
G° = -2880 kJ/mole
• This is a spontaneous reaction, but we have
all seen sugar sitting on a tabletop in the
open air
– The sugar doesn’t spontaneously combust because the Activation
energy for the process is too high
Enzyme Catalyzed Reactions
• An enzyme decreases
the activation energy
barrier
• This allows the
reaction to proceed at
an appreciable rate
• By lowering the
thermodynamic
barrier, we can greatly
increase the rate
(kinetics) of the
reaction
The Effect of Temperature
• Most reaction
rates increase as
the temperature
increases
• For nearly every
enzyme, this is
true up to a
point…
– Thermal
denaturation
Kinetics: Expressing/Describing the
Rate of a Reaction
(See the excellent review link on the “Useful Links” page)
• A rate is traditionally expressed as the:
change in concentration or amount of a substance
time
• The substance can be a reactant or a product
 – If it is a reactant, the rate will have a ____ sign
A + B --> P
-[A] -[B] [P]
Rate =


t
t
t
Rate refers to rate of product
formation or rate of reactant
disappearance
Kinetics: Rate Equation
A + B --> P
The rate of the reaction is slightly different
than strictly looking at the rate of
disappearance of reactant or formation
of product
Rate of Reaction = k[A]f[B]g
– where f and g must be empirically determined
Reaction Orders
• The reaction order is an indicator of the
details of the reaction mechanism
– How many molecules are involved in the
reaction
– The role of the catalyst in the reaction
– Specifics of the system
• We’ll only be concerned with 0th, 1st and
2nd order reactions.
Zero Order Reactions
A --> B
Reaction rate = k[A]0
• The reaction rate is independent of the
substrate concentration
– Catalyst concentration is what matters in this case
• For enzyme catalyzed reactions, we may see
such a reaction order when the substrate
concentration is VERY high and the enzyme
molecules are completely saturated
• The cars over the bridge analogy (Six lanes down to 2)
1st Order Reactions
A --> P
Reaction rate = k[A]1
• This reaction is first order with respect
to reactant A
– What does this mean?
2nd Order Reactions
Glycogenn + Pi --> Glucose-1-Phosphate + Glycogenn-1
Reaction rate = k[Glycogenn]1[Pi]1
• Both glycogen AND Pi have a role in the
reaction
– The reaction is first order with respect to each
reactant, but it is a Second order reaction overall
– What does this mean?
• Change either reactant concentration and what would
happen to the rate?
Section 6.4: Enzyme-Substrate Binding
•
There are 3 major players to consider when
evaluating enzyme-catalyzed reactions:
1. The enzyme: The catalyst
2. Substrate: The Reactant / Starting Material
3. Product: The product of the reaction / What is
released from the enzyme after the reaction
•
For the rest of the chapter, we’re going to
focus on the interplay between these
species
Scheme of an Enzyme-Catalyzed Reaction
1. The Enzyme BINDS the substrate,
forming the E·S complex
(Note the terminology: Big Thing BINDS Little Thing, not the other way around)
2. The E·S complex forms the Transition
State (EX‡) species, which then
rapidly forms the product
3. The Product rapidly dissociates from
the enzyme, regenerating the catalyst
Formation of the E·S Complex
• The substrate is bound to the active site of the enzyme
– Usually (but not always) by covalent means
• There are two models that have been created to
describe this process:
Formation of the E·S Complex:
Lock and Key model
• The active site has a complementary shape to the
substrate.
– It exactly fits the substrate
– Doesn’t take into account Conformational Flexibility!
– Has fallen into disfavor due to its simplicity
Formation of the E·S Complex:
Induced Fit model
• The active site changes shape are the substrate binds,
thereby allowing a low energy complex to form.
• This model allows for substrate variability
– AD H will react with several aliphatic alcohols
– Cytochrome P450s can handle various drugs
• There is a limit: Splenda vs Sucrose
Formation of the E·S Complex
• What would happen if the E·S complex was perfect?
– Think about the energy in the diagram
– What would EA be?
Formation of the Transition State EX‡
and Product Release
• The bond substrate must adopt a
conformation of the transition state
– By this we mean A LOT of things…
• The substrate and the reactive residues of the enzyme
are in close proximity
• Partial bonds are forming, other bondsare breaking,
atoms are shifting around
• Proximity and Orientation determine rate
• Due to its high energy level, the transition
state is just as its name implies: Transitory
• As soon as the transition state complex is
formed, the product is released
Let’s Look at 2 Different Enzyme
Catalyzed Reactions
1. Chymotrypsin
• Catalyzes the
hydrolysis of peptide
bonds AND ester
bonds
– Peptide hydrolysis is its
primary function
•
We can take
advantage of the ester
hydrolysis function to
monitor the Activity of
the enzyme using pnitrophenylesters
2. Aspartate
transcarbamoylase
• The enzyme catalyzes
the formation of
carbamoyl aspartate
from carbamoyl
phosphate and
aspartate
• We can monitor the
activity of the enzyme
directly by
spectrophotometry
Chymotrypsin-catalyzed Ester
Hydrolysis
•
•
At low [Substrate], the
activity is low
As more substrate is added,
the rate increases until it
reaches a maximum
Aspartate Transcarbamoylase
•
•
As the [Substrate] increases, the
activity does not increase as much
until a critical concentration is
reached
The sigmoidal curve seen for this
reaction is indicative of something
else going on…
ALLOSTERY!
Allostery
• Allostery is defined as: “Of or involving a change in
the shape of and activity of an enzyme that results from
molecular binding with a regulatory substance at a site other
than the enzymatically active one.”
Huh?
• When a substrate (or an inhibitor) binds to the
enzyme somewhere OTHER than the active
site, a conformational change may occur that
allows more substrate to bind (or less) at
other subunits in the quaternary structure and
increase (or decrease) the activity.
Allostery
Positively
allosterically
regulated by ATP and
negatively by CTP
•ATP binds to the
R subunits and
causes the C
subunits to open
up and bind
substrate
•CTP causes the
C subunits to
close up
The sigmoidal curve seen for ATCase is an example of
Positive Cooperativity caused by allostery

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