LECTURE 13: CATALYSIS AND KINETICS

Enzyme
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The role of enzymes in facilitating all the actions in keeping the cells alive
There are a number of biochemical reactions, that uncatalyzed, but never happen at a physiologically
relevant timescale
o Meaning seconds or minutes
How does an enzyme accelerate a chemical reaction?
o It lowers the energy barrier
o Well how does an enzyme lower the energy barrier?
It lowers the energy of the transition state
o Well what are chemical reactions?
A chemical reaction between molecules A and B
A reaction is a collision between A and B with the right SPEED and
ORIENTATION
As a result new bonds are made/broken
The probability, the likeliness that two molecules collide with the right speed and
orientation, that probability is reflected in the height of the activation barrier
The higher the activation barrier, the less likely it is that under, physiological
conditions, two molecules that need to react will have the right speed and
orientation to effectively undergo a chemical process
If you were an organic chemist how would you speed up a chemical reaction, knowing that the reaction is a
factor of speed and orientation?
o How to speed up a chemical reaction? What would you do?
1. Increase the temperature
If a temperature increases by 10 degrees centigrade, the reaction rate doubles
The kinetic energy of moelcules is increase and thus they move faster
But how to control orientation?
2. Increase the concentration
More molecules per volume, the chances that one of those molecules is in the
right orientation si increase
o Now, what can a cell control?
Can the cell raise the temperature?
Temperature is a factor nature cant, doesnt want to change
Human body temperature is stuck at 37 degrees centigrade, we can tolerate two or three
degrees
o What about concentration?
How can nature control concentration? ENZYMES
How does the enzyme affect the concentration?
IT BINDS! If an enzyme has a high binding affinity, then the enzyme can grab a
substrate out of solution and hold on to it
If it binds to different reactants, it can increase the EFFECTIVE
CONCENTRATION
Because they can bind their reactants, they effectively increase the local
concentration, it artificially raises the concentration
Metal stick/pencil
o Catalyzing the breaking of the pencil
What really happened?
o How did he break the linear pencil
Introduced energy by bending the pencil
Bent it to the point right before the pencil broke

If you think about a chemical reaction, the enzyme can help the substrate/reagenet undergo
conformational changes that eventually lead to the point right before the pencil break
That point is referred to as the transition state
That point is where the pencil, in its bent form, has the highest state of energy
o When its in the transition state it has 2 options
1. Return to its original conformation, a nonproductive process
2. To go down the otherside, and break
o The transition state is the arrangement of the reactants in such a state/orientation that they either
go back where they come from or they form products
It is a high energy state and inherently short lived
When people thought about how enzymes increase the effective concentration, they thought enzymes
accelerate a reactions simply by holding on to the substrate and increasing the effective concentration
o But then they realized that it doesnt affect the substrate reaching the transition state
o It increases the concentration but it doesnt help the substrate reach the transition state
The enzyme doesnt just bind the substrate, it has SHAPE COMPLEMENTARITY
o The enzyme initially holds to the substrate, but the shape of the active site is in the shape of the
transition state
o As the substrate moves up the reaction state, as it assumes the transition state, shape
complementarity gets better and better
o Whats the driving force for the substrate changing conformation?
It makes different contacts with the enzyme, what does that mean?
As the substrate bends itself to the shape of the active site, it squeezes out the water
Decreasing the entropic penalty resulting from the hydrophobic effect
As the substrate binds itself, it starts interacting more with the enzyme active site forming
hydrogen bonding interactions, electrostatic interactions
They all stabilize the substrate in the bent conformation
The activation energy of the transition state comes down
The instability of the transition state conformation is partially compensated by
being able to establish additional interactions
At this point there is a 50-50 chance it creates the products or it goes back to the
starting point
o Enzyme tricks the substrate into holding on, increasing its effective concentration, then through
shape complementarity it makes the substrate assume structural orientation that help the reaction
to proceed
In reality, enzymes/proteins are not rigid structure, they are dynamic
o An enzyme that can assume a conformation where it effectively binds a substrate, the enzyme then
changes it structure and bends the substrate as it goes through the conformational change
o Almost like a mechanical action
They increase concentration, have shape complementarity
o

Carbonic Anhydrase
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Equilibrium reaction between carbonic acid and carbon dioxide

Histidine side/imidazole moiety and zinc ion
What exactly happens?
o Initially a water binding to zinc
What does this do? It holds the water molecule in position for a longer period of time
Increases the effective concentration of water?
The kelation of water molecule to the zinc, one of the protons of the water is pointing
straight to imidazole of histidine side chain
Imidazole acts as a general base, it begins pulling the proton

Zinc increase the concentration but also holds the water in the correct geometry and
distance for the imidazole to pull the proton, thus increasing the nucleophilicity of the
water, of the oxygen, making it more reactive
The CO2 is on top, and held in such a position to have a successful bond formation
o These all accelerate the process
o Has a functional/shape complementarity
Effectively binding the water
The active site is shaped so that it has a high affinity for water and CO2
o It facilitates the process by pre-orienting the substrate
It makes sure the water and CO2 are held in the right orientation relative to one another
So the nucleophile only sees one electrophile, the CO2
o Activation of nucleophiles and electrophiles
In this case the activation of a nucleophile
o Metal ion/cofactor assistance
The zinc helps with the orientation and nucleophilic activation
o General acid/base catalysis
There is a general base, the imidazole residue, it pools the proton toward itself
These are all factors that contribute to catalysis

Nucleoside Kinase
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Responsible for the binding of ATP to a nucleobase(in this case thymidine)

Facilitates phosphorotransfer/phosphorylation of thymidine to thymidine monophosphate
Magnesium metal ion kelated in active site and zinc, but zinc does not contribute to reaction
The active site:
o Number of resides that hold the reactants in position
Binding of two substrates is in such as way that the polyphosphate tail of ATP points
directly to the hydroxyl group of thymidine, which is supposed receive the phosphate
group
Shape of active site is such that it automatically positions the substrates in such a way
that the parts of the molecules where the chemistry takes place are put in close proximity
to each other
o What else occurs? An asparagine/glutamine (amide) side chain of an amino acid residue forms
hydrogen bonding interactions with pyrimidine ring of thymidine
How do these interactions contribute to catalysis?
Its like a hand holding on to the substrate
It holds the thymidine in a particular orientation
o What else? The magnesium kelate the phosphate groups, like the amide side chain
It holds the phosphate groups of the ATP molecule in place
Theres also an additional effect on the poly phosphate: it increases the partial positive
charge, pull electron density from polyphosphates toward itself, and has an inductive
effect
It increases the electrophilicity, the partial positive charge on the last
phosphorous group
Why is this important? If you create a better electrophile there must also be a
nucleophile on the other end
o The 5 hydroxyl group, in proximity of the phosphate, of the thymidine molecule is the
nucleophile
Also hydrogen bonds with carboxylate group
Hydrogen on hydroxyl group gets pulled towards carboxylate and creates a nucleophile
on the 5 prime sugar of the nucleoside
Good nucleophile and good electrophile facing each other
o Shape complementarity/pre-orientation of substrates, function of the active site environment

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Cofactor assistance: magnesium helps position but also assists in reaction by increasing
electrophilicity of phosphorous side chain
Nucleophilic activation/general base catalysis: carboxylic side chain, creates better nucleophile by
abstracting a proton from hydroxyl group, acts as a general base
Pi-stacking: tyrosine under the pyrimidine system of thymidine substrate, helps to positioning of
one of the two substrates.
One carboxylic acid acts as a general base, deprotonates the 5 hydroxyl group, what about other
carboxylic acid group?
Why isnt that carboxylic acid a general base? Does it abstract a proton from the other
hydroxyl group?
Why does the phosphate go to the 5 and not the 3 position?
PROXIMITY!
If you put a hamburger in front of Dr. Lutz, who will get to the hamburger first,
Dr. Lutz or Jenny?
o Jenny is equally hungry but Dr. Lutz is closer to the hamburger
The way the enzyme holds the two substrates relative to one another, the 3
hydroxyl group is too far away from the electrophilic phosphate groups
Orientation is very important, that is what facilitates the chemical reaction
Organic chemists use protection groups to stop one hydroxyl group from
reacting
Nature doesnt do protection groups, it uses regioselectivity
By positioning the substrate in a certain it can choose which groups react

Peptide Hydrolysis by Serine Protease

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Proteases are enzymes that cleave peptide bonds

o Uncatalyzed takes about 400 years, half-life
o Proteases are responsible for accelerating this reaction
How does it do this?
o The key is the catalytic triad in the enzymes active site
o What is the catalytic triad?
It consists of three amino acid side chains: a serine on the right, a histidine in the middle,
and a aspartate on the left
Those three residues are oriented relative to one another in such a way that the aspartate,
which is deprotonated under physiological conditions, pulls a proton on the imidazole
moiety from the histidine towards itself
o What does that do to the histidine sidechain?
Its proton becomes stolen from the aspartate, what does that do?
It becomes partially negative charged, it looks for another residue to steal a proton from
It takes a proton from the hydroxyl group of the nearby serine
o What does that do to the serine?
IT MAKES IT A GOOD NUCLEOPHILE! It can now do nucleophilic attack!
o Where is there a good electrophile?
Turns out that if the protease binds a polypeptide sequence and positions the peptide bond
it wants to cleave in proximity of the nucleophilic serine side chain then nucleophile can
attack the peptide bond and cleave the peptide bond
Exactly what happens in serine proteases
o Substrate is bound in active site, there is a tunnel/cleft where the polypeptide binds to
o The catalytic triad is in close proximity to where the polypeptide sequence is held in position
o What happens next?

The serine residue attacks the carbonyl of the peptide chain, forms a tetrahedral
intermediate, it breaks apart, the peptide bond is cleaved releasing the nitrogen end of the
polypeptide sequence
Second part
A portion of the peptide is now covalently linked to the enzyme, which would destroy the
catalyst
There must be a mechanism to regenerate the enzyme
After the first portion of the peptide is released, a water molecule comes in and interacts
with the serine and histidine
Via general acid-base catalysis, the water molecule is activated, creating a good
nucleophile, which attacks the ester (the acyl intermediate between the peptide and the
enzyme), forming a tetrahedral intermediate, results in regeneration of the enzyme and
release of the second product
Shape/functionality complementarity
Pre-orientation of substrate
Activation of nucleophiles/electrophiles
General acid/base catalysis
We have hundreds of different proteases in our body, why?
Because the cell wants to control where, which peptide bond it cleaves
It can control that by reshaping the peptide binding pocket

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