Mamalapat, Mohamidin K.

8/26/2023
Chem 2065 AY2023-24
The Behavior of Proteins: Enzymes

1. Compare and contrast the properties of ordinary catalysts versus biological catalysts

Ordinary catalysts are substances that increase the rate of chemical reactions.
They typically enhance the rate of reaction by factors of 10 ² to 10 ⁴. In contrast, biological
catalysts, commonly called enzymes, are usually globular proteins, except for some RNAs
(ribozymes) that exhibit catalytic activity. Enzymes are the most efficient catalyst since
they increase the reaction rate by a factor of up to 10 ²⁰, which is higher than the effects
of the ordinary catalyst.
Unlike ordinary catalysts, enzymes convert the substrate into products when it
forms an enzyme-substrate complex. Also, enzymes exhibit specificity wherein it only acts
on compounds and not the others. Furthermore, they can be fine-tuned by regulatory
processes because they can either speed up or slowed down depending on the cell's
metabolic needs.

2. Review the difference between thermodynamics and kinetics (Is the reaction
"spontaneous"? Is the reaction "fast"?)

Thermodynamic centers on the difference between the energies of the products

and the reactants of a given reaction. This energy change is expressed as the standard
free energy change. That being said, this describes the behavior and the equilibrium
composition of a system. The thermodynamic favorability depends on the free energy
change (∆Gº), wherein a negative ∆Gº' would indicate a spontaneous reaction while a
positive ∆Gº' indicated a nonspontaneous reaction. It should be taken note that a
spontaneous reaction does not have any relationship with the reaction rate, meaning that
the reaction could either be quick or slow.

In contrast, kinetics focuses on the reaction rate at which a specific process will
happen and the pathway by which it will occur. Hence, enzymes that speed up reactions
cannot alter the equilibrium constant or the free energy change because the reaction rate
is dependent on the activation energy, indicating that the activation energy for the
uncatalyzed reaction is higher than that for a catalyzed reaction. Thus, lower activation
energy indicates a faster reaction and vice versa.

With that being stated, a system can be considered thermodynamically favorable

if its reaction is spontaneous (-∆Gº), and its reaction is considered fast if it has lower
activation energy.

3. Differentiate between the progress of ordinary reactions and of enzymatic reactions using
the free energy profile (reactants, transition state, products, progress of reaction,
free energy, activation energy (or Ea), effect of temperature)

In ordinary reactions, as seen in the illustration at the left, the graph plots the
energies for an exergonic reaction wherein there is a release of free energy indicating a
forward reaction. At the maximum of the curve connecting the reactants and the product
lies the transition state where it could go back to being reactants or proceed into as
 products when old bonds break and new bonds formed. The activation energy to bring the
reactants to the transition state is comparatively higher because of the lack of enzymes.
Also, the free energy change is negative due to the downtrend slope going to the products.
This implies that the reaction is spontaneous. Furthermore, when the reactions are
introduced with increasing temperature, the rate of reaction increases since particles can
collide more frequently, enabling the reaction to occur faster. Delta G = free energy change
is same since exergonic

Conversely, in an enzymatic reaction, as seen in the illustration above, it is still an

exergonic reaction, and the free energy change remains unchanged. However, when the
catalyst was introduced, it involved an alternative mechanism wherein the activation
energy was lowered. This implies that the reaction occurs faster compared to an ordinary
reaction (without an enzyme). Also, as the temperature increases, the reaction rate
increases. However, since proteins are peptide chains, the intermolecular forces that hold
them together can be overcome by excessive temperatures leading to the denaturing of
the protein. Above this temperature, adding more heat will continually denature more
enzymes and slows the reaction.

4. Correlate rate of reaction with activation energy

The rate of reaction has an inverse relationship with the activation energy, wherein
the higher the activation energy, the slower the reaction will become. The reason for this
is that molecules require a certain amount of energy to undergo chemical transformation.

In order to lower the activation energy of the reaction, enzymes are added, wherein
it involves an alternative mechanism. Since the activation energy is now lowered, the
reaction can now proceed faster.

5. Review the basics of chemical kinetics: (rates of reactions, monitoring rates of reactions,
rate law, rate constant, first-order reactions, second-order reactions, zero-order reactions,
overall order of reaction (in reality, not very practical), effect of changing the concentration
of reactant to the rate of the reaction)

In the generic reactions, its reaction is given by the

aA + bB --> products, wherein the small letters represent the coefficients, and capital
letters are for the molecules' generic name.

For the rate of reaction, which is the speed at which the chemical reaction occurs, is
defined by the equation called the rate law:
 In the rate law, the rate will be proportional to the concentration A raised to x and
concentration B raised to y. It should be taken into consideration that the x and y are
orders of the reaction and are determined experimentally.

In addition, the reaction rate depends on the activation energy of a particular

reaction. With the presence of enzymes, the rate of reaction increases for the enzymes
provide an alternative mechanism where the activation energy is lowered. It should be
taken note that the enzymes cannot affect the free energy change.

Moreover, the order of reaction refers to the dependence of the rate on the
concentration of the species. In the first-order reaction, the rate is dependent on the first
power of the concentration of a single reactant. Thus, the rate of reaction is directly
proportional to the concentration of the reactant.

While in the second-order reaction, the rate depends on the product of the
concentration of two reactants. Thus, the rate of reaction is proportional to the square of
the concentration of the reactant. An example of this is when the concentration is doubled,
the rate will be quadrupled. Conversely, in a zero-order reaction, the reaction proceeds at
a constant rate, independent of the concentration of the reactant.

Based on the illustration above, the overall order of the reaction is not very practical
since the x and y do not necessarily correspond to the coefficients of the reactants in the
balanced chemical equation.

Lastly, in a reversible reaction, the reaction proceeds in both the forward and
backward directions. When the forward reaction rate is equal to the rate of the reverse
reaction, there is an equilibrium. The equilibrium is defined by the equilibrium constant
(Keq):

6. Describe the overall mechanism of substrate binding on to the active site of enzymes
In an enzyme-catalyzed reaction, the substrate binds to the active site of the
enzymes, where it will be bound by noncovalent forces such as hydrogen bonding,
electrostatic attractions, or Van der Waals attractions. The reason for the binding of the
substrate to the enzyme is due to the highly specific interaction between the substrate and
the side chains and backbone groups of the amino acids making up the whole active site.
Once the substrate binds to the enzyme, it forms an enzyme-substrate complex that leads
to the formation of the transition-state species and eventually forms the product.

7. Differentiate between the lock-and-key model and the induced-fit model of substrate
binding
There are two models to describe the formation of the enzyme-substrate complex.
First is the lock-and-key model wherein a substrate binds to the active site of the enzyme
such that the active site and the substrate exactly match each other in shape
(complementary). While in the induced-fit model, binding of the substrate induces a
 change in conformation of the enzyme that results in a complementary fit. Whether it is
lock and key or induced fit, the formation of the product will occur once the enzyme-
substrate complex is formed.

8. Correlate the free energy of the system (reactants/transition state/product) as the

enzymatic reaction progresses

In the enzymatic reaction, for the reactants to undergo the transition state,
activation energy is required. Once the transition state is reached, it could either go back
to being a reactant or proceed into the products.

It should be taken note that in reverse reactions, the products can be converted
back to the transition and then go back to being reactants, but the activation energy
needed for it to go back to the transition state is higher than the forward reaction (reactants
- transition state- products).

With the presence of enzymes, it increases the reaction rate by providing an

alternative mechanism; hence the activation energy required is lowered. With this, the
reaction proceeds faster compared to an uncatalyzed reaction. However, in the enzymatic
reaction, the free energy change is not affected by the enzymes.

9. Explain why the formation of the enzyme-substrate complex (ES) is a necessary step in
the formation of the product in enzymatic reactions

The enzyme and the substrate will have to first interact with each other, so they
will need small activation energy; hence the binding will happen quickly, and it will easily
form an ES complex. The ES is a necessary step since it is needed for the ES to proceed
to the enzyme-transition state complex before it forms the product. After the formation of
the product, it will be released since it does not have a complementary shape to the active
site of the enzymes, thereby resulting in an unstable interaction. With that, the enzyme
can now attract another substrate, and the cycle again continues.

10. Explain the details in the individual steps in the Michaelis-Menten mechanism of
converting substrates into products

In the Michaelis-Menten mechanism, the enzyme and substrate are in equilibrium with the
ES, and when this complex is formed, then the product will also be formed. The mechanism
of the binding of the substrate to the enzyme is governed by the rate constant k₁ to form the
ES, but since it is in equilibrium, it is possible to have a reverse reaction wherein the ES can
revert back to being an enzyme (E) and substrate (S). This mechanism of reverting the ES to
E+S is governed by the rate constant k₋₁.
In the next step, the ES is governed by the constant rate k₂ to form the product. Once the
product is formed, it will eventually be released from the enzyme due to unstable interactions.

11. Cite the implications of the initial velocity of reactions

In measuring the rate/velocity of an enzymatic reaction at varying substrate

concentrations, it is apparent that the velocity depends on the substrate concentration.
Hence, the initial velocity of the reaction is measured so that we can be assured that the
product is not converted to a substrate to any appreciable extent. This means that the
amount of product made will be measured at the beginning of the reaction as it is
increasing linearly. Lastly, it should be taken note that the calculations involved in enzyme
kinetics assume that the velocity measurement is the initial velocity.

12. Generate a typical kinetic plot of enzymatic reactions (reaction velocity vs. substrate
concentration, constant enzyme concentration)

In this graph, the rate and the observed kinetics of an enzymatic reaction depend
on the substrate concentration. In the lower region of the curve, the reaction is in first
order; this indicates that the velocity depends on the substrate concentration. While in the
upper region of the curve, the reaction is in zero-order, implying that the velocity is
independent of the concentration of the substrate. However, at infinite substrate
concentration, the reaction would proceed at its maximum velocity (Vₘₐₓ).

13. Understand the derivation of the Michaelis-Menten equation based on substrate

concentration levels and the assumption of a 'steady-state' condition

The rate of the formation of the ES is given by the equation where the Δ [ES]/ Δ t
means the change in the concentration of the complex.

As the ES breaks, it breaks down into two reactions which could either be returning
to enzyme and substrate by giving rise to the product and releasing the enzyme. The rate
of the breakdown of the ES is given by this equation
 In the equation above, the negative sign in the term indicates that the concentration
of the complex decreases as the complex breaks down. The k₋₁is the rate constant for the
dissociation of the complex to regenerate the enzyme and substrate, while the k₂ is the
rate constant for the reaction of the complex to form the product and release the enzyme.

Assuming that the steady-state is achieved, at some point in the reaction, the
concentration of the ES is constant; hence, the rate of the formation and of the ES will be
equal to the rate of the breakdown of the ES. Also, under this state, the concentration of
the free enzyme is the total amount of the enzyme concentration minus the ES.

By substituting the concentration of the free enzyme to the equation at steady state
and by collecting all rate constants in one term, we can get the Michaelis constant.

14. Understand the significance of the Michaelis constant, KM (K-subscript 'M')

The Michaelis constant (Kₘ) equals the concentration of substrate when 50% of
the enzyme active sites are occupied by a substrate. The Kₘ is simply the dissociation
 constant for the ES complex on the assumption that k₋₁ >> k₂. This constant measures
how tightly the substrate is bonded to an enzyme. The greater the value of the Kₘ, the
lower the affinity for substrate, indicating that the substrate is less tightly bound to the
enzyme. While the lower the value for Kₘ, the higher the affinity for the substrate indicating
the substrate is tight bound to the enzyme.

Also, the Kₘ can also be used as an indicator if the enzyme is activated or not. If
the substrate concentration is less than the Kₘ, then the enzyme is not activated. While if
the substrate concentration is greater than Kₘ, then the enzyme is activated.

15. Understand the implications when the experimental conditions are such that [S] = KM

In an experimental condition, when the substrate concentration is equal to the

Michaelis constant, the rate of reaction is half its maximum value. This can be seen in the
Michaelis-Menten equation.

16. Graphically derive the value of KM from a Michaelis-Menten Plot

Based on the graph, to graphically derive the value of the Michaelis constant, the
maximum velocity (Vₘₐₓ) and half of the Vmax must be determined first. After that, draw a
line from the point of the Vₘₐₓ/2 until you find the point on the graph that corresponds to it.
Once that is determined, the substrate concentration at that point will give the value of the
Kₘ. The reason for this is that the Kₘis also the substrate concentration at which the
enzymes operate at half of its maximum velocity.
17. Explain the complications or the low-reliability in determining the value of KM using
graphical methods from a Michaelis-Menten Plot

Using the Michaelis-Menten Plot, it is difficult to determine the Vₘₐₓ using a

hyperbolic equation because it is an asymptote, and the value is never reached with any
finite substrate concentration that we could use in the lab. Hence, this also made it difficult
to determine the Kₘ of the enzyme.

18. Be aware of other enzyme mechanisms aside from a simple 'one substrate-one product'
system (ordered, random, ping-pong) [We will not go deeper into this]

The involvement of multiple substrates in an enzyme-catalyzed reaction leads to

a more complex order of events. The most common mechanisms are the ordered
mechanism, the random mechanism, and the ping-pong mechanism.

In an ordered mechanism, there is specified order that substrates must bind, and
the products must be released. It can be seen in the equation below.

In a random mechanism, either one of the substrates can bind first, and either of
the products can be released first. This is apparent in the equation below.

With a ping-pong mechanism, one substrate binds to the enzyme and reacts,
forming a modified version of the enzyme (E') plus the product, P. Once the product is
released, the second substrate binds.

19. Determine the value of KM and Vmax (V -subscript 'max') from a Lineweaver-Burk
double-reciprocal plot (or simply Lineweaver-Burk plot)
 In the Lineweaver-Burk double reciprocal plot, the formula of the Michaelis-Menten
equation, which is a hyperbola, is transformed into an equation for a straight line by taking
the reciprocal of both sides.

Now the equation has the form of a straight line which is y= mx +b. In this new
equation, the slope of the line is Kₘ/Vₘₐₓ, and the y-intercept is 1/Vₘₐₓ. The x intercept is
-1/Kₘ. With this, we can determine the value of the Kₘ and Vₘₐₓ. To determine the Vₘₐₓ,
we can estimate the value of the y intercept and take its reciprocal. At the same time, the
KM can be determined by taking the negative reciprocal of the x intercept or by substituting
the Vₘₐₓ in the slope formula to find the Kₘ.

20. Explain the significance of the KM value to the efficiency of the enzyme

The Michaelis constant (Kₘ) equals the concentration of substrate when 50% of
the enzyme active sites are occupied by a substrate. The Kₘ is simply the dissociation
constant for the ES complex on the assumption that k₋₁>> k₂. This constant measures
how tightly the substrate is bonded to an enzyme. The greater the value of the Kₘ, the
lower the affinity for substrate, indicating that the substrate is less tightly bound to the
enzyme. While the lower the value for Kₘ, the higher the affinity for the substrate indicating
the substrate is tight bound to the enzyme.

On another note, the Kₘ can be used as an indicator whether the enzyme is

activated or not. If the substrate concentration is less than the Kₘ, then the enzyme is not
activated. While if the substrate concentration is greater than Kₘ, then the enzyme is
activated.

21. Explain the significance of the Vmax value to the efficiency of the enzyme (related term
'turnover number')

The Vₘₐₓ is related to the turnover number of a particular enzyme. It is also related
to the catalytic constant, k ₂, and this is also referred to as kcₐₜ.

The turnover number is the number of moles of substrates that react per second
per mole of enzyme. The kcₐₜ serves as an indicator as a measure of enzyme efficiency.
The higher the value of the kcₐₜ, the efficient the enzyme is, while the lower the value of
the kcₐₜ, the slower/less efficient the enzyme is.
22. Using the examples in Table 6.2, correlate the significance of the functions of the listed
enzymes to their reported KM and Vmax values [Why do some enzymes act fast? Why do
some enzymes act slowly?]

Enzymes are very specific, and their efficiency is measured by the turnover
number. The higher the turnover number, the higher the Kₘ; thus, it entails that the
enzyme is very efficient, and it occurs fast. In the table, some enzymes act fast due to the
threat that a particular substance poses, such as in the case of Hydrogen peroxide (H₂0₂),
which is toxic to the cell. The turnover number of catalase is high so that it can quickly
break the H₂0 ₂ down. While in some cases, enzymes act slowly, such as in the case of
Chymotrypsin, wherein its turnover number is relatively low because there is not much
threat to the body if the proteins are not digested immediately. This implies that the speed
of the enzyme depends on the metabolic needs of the cell.

23. Differentiate between the behavior of chymotrypsin and of ATCase in terms of the plots of
their initial velocity versus substrate concentration

The comparison between the behaviors of the chymotrypsin and of the ATCase is
related to the oxygen-binding behaviors of myoglobin and hemoglobin. In the case of the
ATCase, it is considered an allosteric protein that is similar to hemoglobin, while the
chymotrypsin and myoglobin are not.

The chymotrypsin is a nonallosteric enzyme, and it exhibits hyperbolic kinetics,

which is apparent in its slope.

In contrast, the ATCase having multiple proteins exhibits cooperative effects such
that the binding of one molecule of substrate affects the binding of the next molecule of
substrate. As seen in its graph, it exhibits sigmoidal kinetics.

24. Evaluate whether the kinetics of an enzymatic reaction falls under the Michaelis-Menten
kinetics assumptions or conditions
 Michaelis-Menten kinetics describes the behavior of many enzymes. In this case,
it follows a 1:1 correspondence wherein the substrate binds to an enzyme to form an ES
and eventually forms a product. An enzymatic reaction falls under the Michaelis-Menten
kinetics if its curve in the graph is hyperbolic. In the case wherein the curve of the graph
is sigmoidal, or if there are more than two substrates involved, then it does not fall under
the condition of the Michaelis-Menten kinetics.

25. Explain the mechanism of competitive inhibition

In competitive inhibition, the inhibitor binds to the enzyme's active site and blocks
the substrates' access to it. With this, there is competition between the inhibitor and the
substrate for the active site. No product is formed in this mechanism since ES is not
formed, which is a prerequisite for products. As a result, there is a decrease in enzymatic
activity since the presence of an inhibitor reduces the concentration of the enzyme
available to the substrate.

However, the effects of the competitive inhibitor can be overcome by increasing

the concentration of the substrate because whichever has the greater amount (inhibitor or
substrate) has, the higher probability of interacting with the enzyme's active site.

26. Determine whether a given inhibitor is a competitive inhibitor from kinetic data

Using a Lineweaver-Burk plot and comparing the plot of the uninhibited reaction to
the inhibited reaction, we can identify the inhibitor as competitive if the curves intersect on
the y-axis. The reason for this is that both the inhibitor and the substrate are vying for the
same location; hence, they are competing for the active site of the enzyme.
27. Explain the mechanism of non-competitive inhibition

In noncompetitive inhibition, the inhibitor binds to a location other than the

enzyme's active site, and it distorts the active site leading to inhibition of the enzyme
activity. In this type of inhibition, the binding of the inhibitor does not affect the binding of
the substrate. Hence, the substrate can bind just as well to the enzyme (E) as it can to
the enzyme-inhibitor complex (EI). Also, the inhibitor could also bind just as well to E as it
can to ES. However, it should be noted that no product is formed since the product can
only be formed by the ES alone.

On another note, under this mechanism, the Kₘ remains unchanged because the
inhibitor does not interfere with the binding of the substrate to the active site. Unlike
competitive inhibition, under this mechanism, increasing the substrate concentration
cannot overcome the noncompetitive inhibition.

28. Determine whether a given inhibitor is a non-competitive inhibitor from kinetic data

Using a Lineweaver-Burk plot, one can identify the inhibitor as a non-competitive

inhibitor if two lines intersect in the x-axis. Under this mechanism, the y-intercept and the
slope will be changed, but the x-intercept remains unchanged. Furthermore, the Kₘ
remains unchanged since the inhibitor does not affect the binding of the enzyme and the
substrate.
29. Explain the mechanism of uncompetitive inhibition

Under uncompetitive inhibition, the inhibitor binds to the ES but not to the free
enzyme, forming an EIS (Enzyme-Inhibitor-Substrate complex). As a result, the Vmax
decreases because any amount of the inhibitor will tie up some of the ES into the EIS.
Also, the Kₘ decreases because the EIS reduces the concentration of the ES. Thus, by
Le Chatelier's Principle, a shift occurs, stimulating the formation of additional ES.

30. Determine whether a given inhibitor is an uncompetitive inhibitor from kinetic data

Using the Lineweaver-Burk plot, the plot of the uncompetitive inhibitor is parallel to
the uninhibited enzyme. In this case, in determining an uncompetitive inhibitor, its
Lineweaver-Burk plot shows two lines that are parallel to each other. Also, an effect of this
inhibition is that it decreases the Vₘₐₓ and the Kₘ.

31. Account for mixed inhibition types in some enzymes

Under the mixed inhibition, the inhibitor can bind through a mix of the various
mechanisms, as previously stated. Under this mechanism, the inhibitor alters the Kₘ and
Vₘₐₓ, wherein the Kₘ increase while the Vₘₐₓ decreases. Unlike noncompetitive inhibition,
the binding of the substrate or the inhibitor affects the enzyme's binding affinity for the
other. Hence, the binding affinity for the substrate is decreased when the inhibitor is
present and vice versa.
 On another note, in this mechanism, the Lineweaver-Burk plot shows lines for
uninhibited and inhibited reactions intersect in the second quadrant.

32. Account for the effects of irreversible inhibition or of 'suicide substrates'

Under irreversible inhibition, the inhibitor covalently binds to the enzyme

permanently and inactivates it. The inhibitor in this mechanism never lets go its binding to
the enzyme. Thus, permanent changes in the original enzyme cannot be regenerated,
making it no longer able to accept substrate into its active site; thus, no products are
formed.

These molecules that are created for the specific purpose of covalently binding to
the enzyme and permanently inactivating it is called suicide substrates. These molecules
are commonly used in the field of medicine, such as in the case of the antibiotic penicillin
that binds to a specific serine residue of an essential bacterial enzyme. Hence, making
the bacteria unable to make their cell walls, thus killing it.

33. Apply what you learned about enzymes and inhibition in the treatment of AIDS [6D
Biochemical Connections]

In the treatment of the acquired immunodeficiency syndrome (AIDS), a key

strategy in tackling this is through the development of specific inhibitors that selectively
obstruct the enzymes of the human immunodeficiency virus (HIV) that causes AIDS. With
this concept, it could decrease or inactivate the enzymatic activity of the virus.

Three key enzymes are the current targets for AIDS therapy, reverse transcriptase,
integrase, and protease. In the case of HIV, one of the essential target enzymes is the
HIV protease, wherein it is responsible for the production of new virus particles in infected
cells. It catalyzes the processing of viral protein in the infected cells. Without these
proteins, the virus particles cannot be released to cause infection.

In tackling this enzyme, the scientist looked into the structure of the HIV protease,
including its active site. Knowing the structure, the scientist designed and synthesized
compounds to bind to the active site, inactivating the enzyme. Thus, these processes led
to the mass production of these compounds that various pharmaceutical companies’
market.
On another note, the most recent target viral enzyme is the integrase which is
responsible for the virus to copy itself into the host cell. Further developments resulted in
the production of a drug that inhibits the integrase enzyme. That being said, by fully
understanding the concept of enzymes and their inhibition, it is possible to deter the
development or production of infectious diseases or viruses.