CLASS: Fundamentals I 11:00-11:30
DATE: 8/20/2010
PROFESSOR: Deivanayagam
I.
Enzyme Kinetics II
Scribe: Jordan Ridgeway
Proof: Caleb Landrum
Page 1 of 3
What is Kinetic Behavior of Enzymes Catalyzing Bimolecular Reactions? [S36]
a. So far you have seen a very simple situation with one enzyme and one substrate, but in general it’s not so
simple. It gets much more complicated
b. There are many different ways in which enzymes can produce a product
i. Can react with one the first time, then a second one, then a product
ii. Usually there are at least two steps involved – two different substrates involved in enzymatic reactions
c. This can happen in a sequential environment – first one thing binds and gets released, then the second
d. These are called single-displacement or double-displacement
e. Can be subclasses
i. Can be ordered set of reactions or random set of reactions
II. Kinetic Behavior of Enzymes Catalyzing Bimolecular Reactions? [S37]
a. Here is a classic example where there has to be an A and B
b. There are two different substrates that it has to bind
c. Gives rise to the complex A E and B together
d. Two substrates will have to become P and Q – the products
i. Two substrates in, two products out
e. You can see here that it becomes a little more complicated
f. In a single displacement reaction, you can see that this is how the Lineweaver-Burk plot looks
g. If you keep increasing concentration of substrate B, the slope keeps changing
h. Usually Vmax is constant in these reactions – because the enzyme is the same
III. Conversion of AEB to PEQ is the Rate-Limiting Step in Random, Single-Displacement Reactions [S38]
a. If you look at the random formation – A randomly binds to E – gives rise to AE
b. Similarly B randomly binds to the enzyme
c. These two combine to give rise to AEB and then PEQ
d. This has to dissociate to EP and EQ
e. In this case you can see that the whole step is limited by formation of AEB and PEQ
f. only the AEB complex can convert to PEQ.
g. If there is too much AE and too little EB, then this limits the entire reaction
h. The limiting step is formation of AEB
i. This is the random way in which reactions proceed
IV. Creatine Kinase Acts by a Random, Single-Displacement Mechanism [S39]
a. Creatine Kinase reacts by single displacement mechanism
b. ATP binds to the enzyme as well as creatine, (either can bind first)
c. Only when that complex forms can it create creatine phosphate
V. In an Ordered, Single-Displacement Reaction, The Leading Substrate Must Bind First [S40]
a. In an ordered reaction, the leading substrate has to bind first
b. A has to bind first, then AE binds to B -> AEB
c. Q is released first and then P
d. In these reactions, the limiting step is formation of AE and release of P
i. Only if A binds can the reaction move forward, and only if P is released does the enzyme become free
VI. The Double Displacement “Ping-Pong” Reaction [S41]
a. In this case, the enzyme binds to A and actually modifies the enzyme to a form what we call E’.
b. Then the product, P is released,
c. Then the next substrate binds and becomes Q and the Enzyme is modified back to E
d. The enzyme releases Q and the enzyme is free again
e. The limiting steps are at A and Q and P and B
i. If A is not bound it does not give rise to the modified enzyme E’
ii. If P is not released, then the enzyme gets stuck
f. These are actually not intermediate reactions, but are intermediate steps in the kinetic reaction
i. If you think of this as step one, then the later intermediate steps is where most of the inhibitors are
designed – in the intermediate states.
ii. If you trap them somewhere here, then the enzyme becomes non-functional
CLASS: Fundamentals I 11:00-11:30
DATE: 8/20/2010
PROFESSOR: Deivanayagam
Enzyme Kinetics II
Scribe: Jordan Ridgeway
Proof: Caleb Landrum
Page 2 of 3
g. An example of this is insulin which breaks bacterial cell walls by binding to transpeptidase which catalyzes
the reaction synthesizing cell walls
i. Insulin binds covalently to the active site of transpeptidase, stopping the enzyme at that point
ii. Also called a Suicide Inhibitor
VII. The Double Displacement “Ping-Pong” Reaction [S42]
a. This is a Double-Displacement “Ping-Pong” reaction
b. These are generally parallel lines
c. If you keep adding substrate B and you get parallel lines on Lineweaver-Burk plot, then you know that you
probably have a double displacement reaction
VIII. Glutamate: aspartate Aminotransferase [S43]
a. A good example of double displacement reaction is the Glutamate: aspartate Aminotransferase reaction
b. From here you know that an amino acid is being transferred
c. You can see that one form is converted to another form which is the E’ form and that starts the release of
alpha ketoglutarate and then the formation of Aspartate

We can view and understand how enzymes work with multiple substrates and specific mechanisms by looking at
these plots (kinetics).
IX. How Can Enzymes Be So Specific? [S44]
a. How can enzymes be so specific? I’m a structural biologist – I look at crystal structures and try to figure out
how the mechanisms happen. If you look at the active sites, you can find residues and try to model
substrates to bind to the active sites
b. It is like a “lock and key” - if other things can bind to active site, it can be a problem
i. Some people thought that because enzymes are so specific, that it had to be a “lock and key” mechanism
ii. In other terms they said the active site was so rigid that the substrate had to be exactly right in its
conformation to fit and bind into the pocket
iii. That was the theory; however, over time, we began to figure out that the enzyme’s active site is mobile
1. In most cases, there are histidines in the active site usually in multiple conformations
a. We call them side-chain rotamers
b. They can be in many different positions
2. Once the substrate comes and binds, it will lock into position and proceed with the reactions
3. That, in a simple way, is called the “induced fit”
iv. Most enzymes, today, people think of as “induced fit” conformations, not “lock and key”
c. Specificity and reactivity are often linked together
i. The general idea is that the active site is not rigid
ii. But in terms of the folds and everything, they are mostly rigid
iii. You can always tell where the active site will be by looking at the model
iv. The fold is always rigid, but the active site is flexible
v. Specificity basically comes from the substrate coming close to the site and the residues in the site align
themselves to create an induced fit
vi. There are some instances where the “lock and key” concept still holds, so that is why we list both of them
here
X. Are All Enzymes Proteins? [S45]
a. Are all Enzymes proteins? NO.
b. There are RNA’s – catalytic RNA’s that do carry out catalytic activity.
i. These molecules are evolutionarily much earlier if you think about it
ii. If you think that they were the first ones to come, they did possess a certain amount of catalytic activity b/c
this was required in order for them to survive
c. There are also antibodies
i. Known for recognition
ii. One half of our immune system would be wiped out if they did not specifically recognize
iii. Generally antibodies are talked about as only having adhesive characteristics
iv. If there is a new “stranger” in your system, antibodies are the first to arrive and they “tag” it then the rest of
the reactions take place
v. But, antibodies also have catalytic activity
1. The antibodies that have catalytic activity are called Abzymes
CLASS: Fundamentals I 11:00-11:30
DATE: 8/20/2010
PROFESSOR: Deivanayagam
Enzyme Kinetics II
Scribe: Jordan Ridgeway
Proof: Caleb Landrum
Page 3 of 3
XI. Is It Possible to Design an Enzyme to Catalyze Any Desired Reaction? [S46]
a. Is it possible to design an enzyme to catalyze any desired reaction? Yes and No
b. YES: If you take a particular enzyme like chymotrypsin, you can modify it several ways
i. You can modify it to be specific only to chymotrypsin,
ii. or you can modify it to react with other similar molecules
c. NO: It is possible to re-engineer them, but if you want to design it from scratch and put particular residues all
over the place, you will win a Nobel Prize. It is not practical because we cannot predict the folding.
** Reminder that Dr. Whikehart emailed the slide with review topics**
[End 17:12]