Mamalapat, Mohamidin K.

9/2/2023
Chem 2065 AY2023-24

The Behavior of Proteins: Enzymes, Mechanisms, and Control

1. Differentiate between allosteric enzymes and 'regular' enzymes that follow Michaelis-
Menten kinetics
Allosteric enzymes are enzymes that have an additional site aside from the active
site that are called allosteric sites. These enzymes typically have multiple subunits, and
they exhibit cooperative effects wherein the binding of one molecule of substrate affects
the binding of the following molecule of substrate. Lastly, it exhibits sigmoidal kinetics,
which is apparent when one plots the velocity and substrate concentration. In contrast,
regular enzymes that follows the Michaelis-Menten kinetics does not exhibit any
cooperative effects, and it has hyperbolic kinetics. Although the Michaelis-Menten model
describes the behavior of most enzymes.

2. Describe the nature of feedback inhibition (a.k.a. end-product inhibition) using the
ATCase reaction as an example
Feedback inhibition is one way to control allosteric enzymes in which the final
product of a series of reactions inhibits the first reaction in the series. An example of this
is the ATCase reaction, wherein the ATCase catalyzes the first step in the reaction, and
the Carbamoyl aspartate undergoes series of reactions to eventually form Cytidine
triphosphate (CTP). Now, the Cytidine triphosphate (CTP) inhibits the ATCase. This
reaction is a good example of how pathways can be controlled to avoid the overproduction
of compounds.

3. Differentiate between feedback inhibition and product inhibition

Feedback inhibition is a case wherein the final product in a series of reactions is
the one that inhibits the first enzyme in the series. While, in product inhibition, the product
that the particular enzyme has catalyze is also the one that inhibits the same enzyme.

4. Illustrate the cooperative behavior of allosteric enzymes using enzyme kinetics (plot of
reaction velocity versus substrate concentration for example) [Note: this section only
wants to highlight that allosteric enzymes can be regulated. Do not worry too much about
the other details within the text.]
 The sigmoidal curve in the model indicates the cooperative behavior of allosteric
enzymes. The behavior of the allosteric enzymes is affected by different substances, such
as in the case of the ATCase, wherein it can be inhibited by the presence of CTP (an
inhibitor), or it can be activated by the presence of ATP (an activator). Furthermore, at low
aspartate concentration, the shape of the activated ATCase is more hyperbolic. Thus, this
could change the behavior of the allosteric enzyme such that it behaves in a way that
obeys the Michaelis-Menten kinetics.

5. Define allosteric effector, homotropic effects, and heterotropic effects

An allosteric effector can be a substrate, inhibitor, or activator that binds to an
allosteric enzyme that changes that allosteric enzyme's quaternary structure. Thus, the
change in quaternary structure changes the behavior of the protein.
On the other hand, homotropic effects are allosteric effects that happen when
several identical molecules are bound to a protein. In contrast, heterotropic effects are
allosteric effects that occur when different substances are bound to a protein.

6. Describe the concerted model of allosteric enzymes

In the concerted model of allosteric enzyme, the protein has two conformations,
specifically the active R (relaxed) conformation that binds substrate tightly and the inactive
T (tight, or taut) conformation, which binds substrate less tightly. The distinguishing feature
of this model that sets it apart from other models is that both subunits change conformation
simultaneously from the inactive T conformation to the active R conformation and vice
versa.

7. Describe how changes in the L (= [T]/[R] ---the text just uses T/R) value affects the
behavior of allosteric enzymes [Does a high L value make it more sigmoidal or
hyperbolic?]
As the L increases and the c is kept constant, the free T form is highly favored,
affecting the allosteric enzyme's behavior, thus, the curve becomes more sigmoidal;
however, it slowly become hyperbolic as the L decreases.

8. Describe how changes in the c (= K(sub R)/K(sub T)) value affects the behavior of
allosteric enzymes [Does a high c value make it more sigmoidal or hyperbolic?]
As the value of c decreases, there is a higher affinity between the substrate and
the R form; hence the shape of the curve becomes more sigmoidal. However, as the value
of c increases, the substrate starts to bind more to the T form, making the curve more
hyperbolic.

9. Describe the sequential model of allosteric enzymes

In the sequential model of allosteric enzymes, the binding of substrate induces the
conformational change from the T form to the R form. The conformational change from
the T to R form in one subunit makes the same conformational change easier in another
subunit. This behavior exhibits cooperative binding.
10. Describe how enzymes can be controlled by covalent modification, specifically by
phosphorylation. (What amino acid residues can be phosphorylated? What changes do
you expect from proteins that are phosphorylated versus non-phosphorylated? What is
the role of kinases in phosphorylation? Does phosphorylation increase or decrease
enzymatic activity?)
Phosphorylation is a biochemical process that requires the addition of phosphate
to a compound. The amino acid residues that can undergo phosphorylation are serine,
threonine both of which have a hydroxyl bonded to an aliphatic group, and a tyrosine which
has a hydroxyl bonded to the aromatic group, phenol. If the source of the phosphate is
from the ATP or similar compounds, they are catalyzed by kinases. The role of the kinases
is that they catalyze the transfer of a phosphate group to some substrate.
The effects of this reaction change the intermolecular force that previously acted
on the amino acid residues, such as in the case of serine, where it has been involved with
hydrogen bonding after as it becomes phosphorylated. Also, there is a change in
conformation after the phosphorylation. Thus, the phosphorylated form is more active
compared to the non-phosphorylated form.
Lastly, there is no way to predict whether the phosphorylation will increase or
decrease the activity of an enzyme since it depends on the situation. In some cases, the
effects of the two opposing enzymes in a system are coordinate. For example, an essential
enzyme in a catabolic (breakdown) pathway may be activated by phosphorylation, while
its counterpart, the anabolic (build-up) pathway, is inhibited by phosphorylation

11. Describe how zymogens are activated by cleavage of peptide bonds. (Use chymotrypsin
as an example)
Zymogens are inactive precursors of the enzyme, and they can be irreversibly
transformed into an active enzyme by breaking peptide bonds and residues. An example
of this is in the case of chymotrypsin.
The inactive precursor of the chymotrypsin, the chymotrypsinogen, is formed in the
pancreas, wherein if it would be in an active form, it could cause damage. Since
chymotrypsin is needed in the small intestine, the peptide bond in Arg 15 of the
chymotrypsinogen is cleaved and is catalyzed by trypsin producing an active π-
chymotrypsin.
The π-chymotrypsin can self-digest, making it able to act on to other π-
chymotrypsin molecules to remove two dipeptide fragments, specifically Ser 14- Arg 15,
and Thr 147- Asn 148 forming α- chymotrypsin. The α-chymotrypsin is now the fully active
enzyme which can hydrolyze proteins found in the digestive tract.

12. Describe the nature and mechanism of the active site (How are the critical amino
acids in the active site identified? How are these critical amino acids arranged in the
active site? What is the mechanism of action of these amino acids?)
Enzymes catalyze a reaction in various ways, and some reactive groups in the
enzymes are required to interact with the substrate. In proteins, the α-carboxyl and the α
-amino group of the amino acids have formed a peptide bond, deeming them no longer
free; hence the side chain reactive groups are involved in the action of the enzyme. The
functional group that could play a catalytic role are the imidazole group (histidine), hydroxyl
group (serine), carboxyl side chains (aspartate and glutamate), the sulfhydryl group
(cysteine), the amino side chain (lysine), and the phenol group (tyrosine). It should be
 noted that when the alpha-carboxyl and the alpha-amino group of the peptide chain are
positioned in the active site, then they can also have a catalytic role.

These critical amino acids are determined through labeling, which is the covalent
modification of a specific residue on an enzyme. Through labeling, you can test whether
a specific residue affects the enzyme or not. For example, in chymotrypsin, TPCK (tosyl-
l-phenylalanine chloromethyl ketone) is used to label His 57, and the phenylalanine part
of TPCK binds to the enzyme due to the specificity for the aromatic amino acid residue at
the active site. The active site histidine reacts since the labeling reagent is the same as
the usual substrate.
The labeling studies show that Ser 195 and the His 57 are required for its activity;
hence Ser 195 and His 57 must be near each other in the active site. The Asp 102 is also
important for its functions to keep the Ser 195 and His 57 in place.

On another note, X-ray crystallography can be used to determine the amino acids
that are located in the active site since by determining the 3-D structure of the enzyme, it
provides evidence that the active-site residues do have a close spatial relationship. Such
as in the chymotrypsin, wherein you can only see the folding of the chymotrypsin
backbone, and it only shows the critical residues that are arranged in the active site. (His
57, Ser, 195, Asp 102- also called as a catalytic triad). These are the only few residues
that are directly involved, and the rest are necessary to provide the correct 3-D
arrangement of the critical residues; thus, the polypeptide chain has to fold to ensure that
the catalytic triad is held together in the active site.

The mode of action of the chymotrypsin is through the hydrolysis of peptide bonds
that are adjacent to aromatic amino acid residues, wherein other residues may be attacked
or hydrolyzed, but at a slower rate. Also, the properties of chymotrypsin enable them to
hydrolyze esters such as the compound p-nitrophenyl acetate. In the mechanism of the
chymotrypsin action, the nucleophilic attack by serine is the main feature in the
mechanism with histidine bonded to serine in the course of the succeeding reaction.

13. Describe the kinetics of chymotrypsin-catalyzed reaction of p-nitrophenyl acetate. (What

does the kinetics imply about the mechanism of action of chymotrypsin?)
The use of model systems provides the essential features of a reaction in a simple
form that is easier to work with than the one found in nature. The typical model compound
is p-nitrophenyl acetate. As seen in the illustration of the kinetics of the chymotrypsin-
catalyzed reaction of p-nitrophenyl acetate, it reveals that there is an initial burst wherein
the p-nitrophenolate is produced first, and then a slower phase wherein the acetate ion is
produced in the latter part. These kinetics imply that the enzyme has two phases.

In the first step, the enzyme(chymotrypsin) and the p-nitrophenyl acetate undergo
a nucleophilic attack producing the acyl-enzyme intermediate and the p-nitrophenolate.
This explains why there is an initial burst of p-nitrophenolate at first and not the acetate
ion since the latter is still bounded in the acyl-enzyme intermediate. While in the second
step, the acyl-enzyme intermediate is hydrolyzed, releasing the acetate and regenerating
the free enzyme.
 14. Describe the individual steps of the mechanism of action of chymotrypsin (What is the
role of the 'catalytic triad' serine-histidine-aspartate in serine proteases?)
In the first stage of the mechanism of action of chymotrypsin, as long as the side
chain is aromatic, a certain section of the polypeptide chain fits into the hydrophobic
pockets. Due to the arrangement of His and Ser, a nucleophilic attack of hydrogen in the
H-bond donated to imidazole occurs, so there is now a nucleophilic attack of serine to the
carbonyl carbon aided by the H-bonding. It should be noted that the Asp 102 is not shown,
but its role is to keep the His and Ser in place.
After the nucleophilic attack of serine to the carbonyl carbon, it results in the formation of
the tetrahedral intermediate, then the migration of electrons occurs, which breaks the
peptide bonds, resulting in the release of amine, and ester is subsequently formed.
In the second stage of the reaction, the water is bonded to the histidine; hence the
water undergoes a nucleophilic attack to the ester. Once again, a tetrahedral intermediate
is formed, and the migration of electrons occurs. Lastly, in the final step of the reaction,
the bond between the serine oxygen and the carbonyl carbon breaks, releasing the
carboxylate portion of the polypeptide chain and regenerating the original enzyme.
It should be noted that serine is hydrogen-bonded to the histidine, and this H-
bonding increased the nucleophilicity of serine in the 1st stage. In contrast, in the second
stage of the reaction, the H-bonding between the water and the histidine increased the
nucleophilicity of the water. This explains the capability of the nucleophile in every part of
the reaction.

15. Describe the expected reaction mechanisms (from your general and organic chemistry)
involved in enzyme mechanisms (nucleophilic substitution, general acid-base
catalysis, metal-ion catalysis (Lewis acid-base catalysis))

Common organic reaction mechanisms such as nucleophilic substitution, general acid-base

catalysis, and metal-ion catalysis have been known to play an important role in enzymatic
reactions.

Where:

Z is a nucleophile X is a leaving group

In a nucleophilic substitution reaction, a nucleophile (electron-rich atom) attacks an electron-

deficient atom. The carbonyl carbon is a common target of nucleophiles. Examples of common
nucleophiles are oxygens of serine, threonine, and tyrosine.

If the rate of the reactions depends solely on the concentration of the R:X, then the
nucleophilic reaction is considered SN1 (substitution nucleophilic unimolecular). In this
mechanism, the slow part of the reaction is the breaking of the bond between the R and the X,
and that the addition of the nucleophile Z occurs very quickly. Thus, the rate of reaction in SN1
follows first-order kinetics.

In contrast, if the nucleophilic attack the R:X while the X is still attached, then both the R:X
concentration and the concentration of: Z will be considered important. This nucleophilic reaction
is called an SN2 reaction (substitution nucleophilic bimolecular), and it follows second-order
kinetics. The difference between SN1 and SN2 is very important since it explains much regarding
the stereospecificity of products formed. In the SN1 reaction, it often leads to a loss of
stereospecificity, for the leaving group is gone before the attacking group enters. Hence, the
attack group can often end up in one of two orientations. While in an SN2 reaction, since the
leaving group is still attached, it forces the nucleophile to attack from a particular side of the bond,
resulting in only one possible stereospecificity in the product.

On another note, in general acid-base catalysis, depends on the donation and the acceptance
of protons. The important functional groups that could either act as acids or bases are
imidazole(histidine), hydroxyl, carboxyl, sulfhydryl, amino, and phenolic side chains. The
donation and acceptance of protons give rise to the breaking of bonds and reformation that
constitute the enzymatic reaction.

In an event wherein the enzyme mechanism involves an amino acid donating hydrogen ions,
then that part of the mechanism is considered general acid catalysis.

General Acid Catalysis

However, if the enzyme mechanism involves an amino acid that takes a hydrogen ion from
one of the substrates, then that part is called the general base catalysis.

General Base Catalysis

 Histidine on the other hand, often takes part in both the acid and base reactions due to its
reactive hydrogen on the imidazole side chain, which dissociates near physiological pH. An
example of this would be in the chymotrypsin mode of action, wherein we saw both acid and base
catalysis by histidine.

Lastly, another form of acid-base catalysis shows a more

general definition of acid and bases. Metal -ions are a form of
catalysis that depends on the Lewis definition of acid as an electron
pair acceptor and a base as an electron-pair donor. There is a
cofactor metal ion incorporated into a protein for this to be possible.
Some enzymes contain metal ions such as Mn²⁺, Mg²⁺, and Zn²⁺,
enabling them to play a role in metal ion catalysis. An example of
this type of behavior is in the involvement of the Zn²⁺ in the
enzymatic activity of carboxypeptidase, wherein this enzyme
catalyzes the hydrolysis of C terminal peptide bonds of proteins.

Reaction catalyzed by carboxypeptidase

16. Describe the importance of co-factors and co-enzymes in reactions that cannot undergo
the mechanisms described in the previous objective. (Cite the roles of NADH/NAD+ in
redox reactions)
Co-factors and coenzymes are nonprotein substances that take part in enzymatic
reactions and are regenerated at the end of the reaction. A few of the essential organic
coenzymes are vitamins and their derivatives, such as B vitamins. The significance of the
co-factors and coenzymes is that they are involved in oxidation-reduction reactions that
provide energy to the organism. Also, others function as group transfer agents in metabolic
processes.
In the case of Nicotinamide adenine dinucleotide (NAD⁺), it is a coenzyme in many
oxidation-reduction reactions. NAD⁺ is composed of primarily three parts which are a
nicotinamide ring, an adenine ring, and two sugar-phosphate groups that are linked
together. The nicotinamide ring contains the site at which oxidation and reduction occur.
The nicotinamide occurs in two forms, the oxidized form (NAD⁺), and it can be reduced by
reducing agent into NADH. In this reaction, the H⁺ is transferred along with the two
electrons and vice versa depending on if the present agent is an oxidizing agent or a
reducing agent.

17. Explain how transition-state analogs (TSA) can help elucidate the mechanisms of
enzymatic reactions
Transition state analogs (TSA) are synthesized compounds with a shape that
mimics the transition state of the substrate. An example of this is the proline racemase
reaction, where the L-proline (substrate) undergoes a racemization reaction to be
converted to D-proline (product) using the racemase enzyme. However, before the
substrate is converted into the product, a planar transition state is formed.
 By using a pyrrole-2-carboxylate (TSA) that is structurally similar to what proline
would look like at its transition state, it could serve as an inhibitor that would bind to the
proline racemase (enzyme). The result in the TSA usage is that it shows that the inhibitor
binds to the enzyme 160 times more strongly than proline does. This indicates that the
TSA can be used to verify the suspected mechanism and structure of the transition state.
Moreover, the TSA could inhibit an enzyme selectively in enzyme-catalyzed reactions by
blocking the enzyme's active site.

18. Describe what abzymes are

Abzymes are antibodies that are produced against a transition-state analog, and these
have catalytic activity similar to a naturally occurring enzyme. Also, abzymes bind strongly
to the transition, thereby increasing the reaction rate. The idea of abzymes first started in
immunogen wherein it would elicit antibodies with catalytic activity if that immunogen
mimicked the transition state of the reaction since antibody, in essence, is just a fake
active site for it is designed to bind to specific molecule on the immunogen. For example,
the N^α -(5'-phosphopyridoxyl)-l-lysine moiety is a transition state analog for the reaction
of an amino acid with pyridoxal 5'-phosphate. When this moiety is attached to a protein
and injected into a host, it functions as an antigen, and the host then produces antibodies
that have catalytic, which we call abzymes. These abzymes are then used to catalyze the
reaction.