LECTURE 14: ENZYME KINETICS

Weve talked about the conceptual underpinning of enzyme catalysis:

o How enzymes accomplish rate acceleration, lowering energy barrier
Shape complementarity
Pre-orientation
Nucleophile and electrophile activation
Think about the events that take place when an enzyme catalyzes a reaction
o Enzyme + Substrate -> black box enzyme + products
o Thats the extent most scientists are working with
o Enzyme catalysis is a series of events
Enzyme can bind Thymidine or ATP first
Whatever 2nd substrate is missing then binds, forms tertiary complex
What are these binding events? What kinetic parameter can describe these binding
events?
The Kd! They are simple binding events.
The tertiary complex then undergoes chemistry, forms second tertiary complex
The products must then leave, most appropriately described by Kd as well
o There must also be multiple conformational changes
o To describe enzyme activity, we can usually stick to simpler, black box versions, where we dont
consider all of the stuff mentioned above
o In addition all reactions are reversible, but we dont care about them because theyre not
productive
Enzymes dont change thermodynamics, they change the KINETICS, they change activations energy NOT
free energy
o Enzymes that can accelerate forward reaction, can also accelerate the reverse reaction
EXCEPT for polymerization of amino acids
This was also an equilibrium, but nature plays this equilibrium by shifting it in
the reaction it wants it to go
Nature hydrolyzes pyrophosphate, one of the products, couples production of
pyrophosphate with second enzymatic reaction
o Energetically highly favorable process, eliminates one of the products,
thereby preventing the reverse reaction
All of these reactions are happening simultaneously, they are all to some degree
coupled, connected
One reactions product is the next reactions substrate, that is the whole point of
metabolism
How to quantitatively describe an enzymatic reaction?
o Hydrolysis of ester, paranitrophenylbutylester
o The ester, under slightly basic conditions, can spontaneously hydrolyze into buteric acid and paranitrophenolate
One of the two products, para-nitrophenolate, is bright yellow
Color associated with product formation
UV spectroscopy can quantify amount of para-nitrophenolate
How do you do this? The absorbance wavelength of yellow color is 450 nanometers, how
do you quantify the yellowness?
Use BEERS LAW, link absorbance to extinction coefficient times concentration
time length of cuvette
Extinction coefficient is characteristic of para-nitrophenolate (10,000 /M/cm)
You can calculate the concentration, the amount of product being formed, for any
absorbance
Following the absorbance over time, you will get a linear relationship

What happens to the reaction if you double the concentration of reactions? How does that affect
the graph?
The slope doubles, tripling the concentration, the slope increases three fold
There is a linear relationship, the higher the substrate concentration the faster the
hydrolysis happens
o Lets add an enzyme, a hydrolase, an esterase that facilitates the reaction, how does it change the
slope?
The enzyme lower the energy barrier, what does lowering the energy barrier do?
It increases the rate of reaction! The slope will be steeper!
o Same experiment with same substrate concentration in presence of enzyme, the slope is steeper
because the enzyme facilitates the chemical reaction.
But it still follows the fundamental concept that doubling the substrate concentration the
slope increases even further.
Will the slope continue to increase by adding substrate?
NO! Why not? Lets think about pencil breaking, what is the limitation?
It takes a finite amount of time to break a pencil, you must pick up a pencil,
have a conformational change, and then drop the pencil.
Theres a finite amount of time required for these actions, that time will be the
limiting step
o If you do this with enzymes, you observe the same effect
As you increase the concentration, the slope slows donw it reaches a Vmax
The enzyme reaches saturation
o Enzyme activity eventually flattens out, you see a bending of the curve, the slope doesnt go down
to 0, it becomes PARALLEL TO THE UNCATALYZED REACTION
The uncatalyzed reaction is running in the background
For biological reacitons, the background reaction is very low, the line would be pretty
much flat
How does that change the curve for the enzyme catalyzed reaction?
Similar to ligand binding curve, it approaches a maximum, flattens out
A typical reaction velocity curve for an enzyme catalyzed reaction
o Initially the rate of reaction, the V0, how many micromoles of product/min is being produces,
increases linearly
o However as substrate increases the enzyme itself, the physical action that is required, starts
slowing down the process overall, you get a hyperbolic curve
o This is a behavior that you can see in the lab if you measure an enzymatic reaction
How is ligand binding different from enzyme reaction velocity curve?
o THEY ARE VERY DIFFERENT
o What are the axes for ligand binding?
X axis: ligand concentration
Y axis: ratio, theta, the fraction of protein that was free vs. bound
o Enzyme kinetics
X axis: substrate concentration
Y axis: NOT A RATIO, it is a velocity, rate of product formation
Michaelis and Menten looked at behavior of enzymatic reaction
o Came up with description of reaction, the michaelis menten equation
o It is a drastic simplification of the reality
If you look at the parameters: they only considered three parameters: K1, K-1, and K2
K1, K-1: the rate by which an enzyme binds a substrate to form the enzyme
substrate complex
o Simple binding event
o

K2: the rate constant for the conversion of the ES complex to enzyme plus
product
o The Michaelis Menten equation assumes a number of things:
1. That there is only one substrate that matters
2. Once you form the ES complex, the chemistry and release of product is all bunched
into the K2 rate constant
o These assumptions can actually hold true
o V0: the velocity by which a chemical reaction proceeds in the presence of an enzyme catalyst
o Vmax: the maximum velocity a reaction can reach, Dr. Lutz in a sea of pencils
o Km: Michaelis Menten constant, different from a dissociation constant,
What is the definition of a Kd?
Speed of forward reaction vs. speed of reverse reaction, K-1/K1
Km, it also factors in the rate of the actual chemical reaction K2, in addition to K-1 and
K1
How can we determine the Vmax and Km?
o The Vmax is pretty straightforward, the more pencils Dr. Lutz has the more likely hell be
reaching his Vmax
As substrate concentration reaches infinity the graph will reach Vmax
Vmax is the maximum velocity at infinitely high substrate concentrations
o Km is the ratio of K1, K-1, and K2
When V0 is half Vmax, you will see the substrate concentration equal Km
The substrate concentration when the enzyme runs at half speed is equal to the michaelis
menten constant
Why is this important?
o If you want measure how fast a drug is metabolized, how long the half life for the drugs is, the
numbers can be very helpful
o It can also quantify the performance of an enzyme with multiple substrates
o Measurement of thymidine kinase with multiple nucleosides and nucleoside analogs:
AZT vs. dU (deoxyuridine)
Which substrate more readily goes into enzyme active site?
AZT, it has a smaller Km, the lower the substrate concentration at which you
reach half maximum speed, the better, it binds quicker
However! What about the Vmax?
dU has a higher Kcat
Although it may not bind as a substrate as quickly, it get turned over much more
quickly
Both Km and Kcat are important, people have come up with catalytic performance or
SPEICFICITY ACTIVITY
Kcat/Km
Why is this important/helpful?
o If you had the perfect enzyme, what would be the preferred Km and
Vmax?
A high Vmax and a small Km
Vmax/Km gives you a very big number
o If you have an enzyme with two different substrates, if you calculate
the Kcat/Km, the bigger the value the better the enzyme
Lineweaver Burk
o Took michaelis menten and inverted it, you obtain a linear relationship
You can plot 1/substrate concentration and 1/reaction velocity, you get a linear
relationship
Allows you measure the slope and y-intercept

1/y-intercept is equal to Vmax, and -1/x-intercept is equal to Km

Hanes Wolf
o Lineweaver Burke but multiplied both sides by substrate concentration
o Whats the advantage?
If you do an analysis by Lineweaver-Burke, the 1/S, 1/substrate concentration, causes a
lot of error
At higher substrate concentration, the lower the value would be, results in a clustering of
data points near 0

Adds a lot of error in analysis, prone to a lot of experimental erro

o Hanes wolf stretches out data points, not prone experimental error