Enzyme-structure and kinetics

Catalysts
Catalysts increase the rate of reaction by providing an alternative pathway which has
a lower activation energy
 Catalysts…
There are two types of catalyst:

1. Heterogeneous – one that is present in a different phase as the reacting molecules.

2. Homogeneous – one that is present in the same phase as the reacting molecules.

1. Heterogeneous Catalysts
 Often we encounter a situation involving a solid catalyst in
contact with gaseous reactants and gaseous products

 Example: catalytic converters in cars. Many industrial catalysts are heterogeneous.

The metals in catalytic converters speed up the conversion of nitrogen oxides and CO
into N2 and CO2 respectively
 Catalysts…
A heterogeneous catalytic reaction has at least four steps in its reaction
mechanism:
1. Adsorption of the reactant(s) onto the surface of the catalyst
2. Activation of the adsorbed reactant(s)
3. Reaction of the adsorbed reactant(s)
4. Diffusion of the product(s) from the surface into the gas or liquid phase (desorption)

Hydrogenation of Ethylene on a Heterogeneous Catalyst

Catalysts…
 Catalysts…
2. Homogeneous catalysts are those that are in the same phase as the reaction
mixture

 In homogeneous catalysis a catalytic reaction in which the catalyst is uniformly

dispersed throughout the reactant mixture to form a solution, the catalyst is in the
same phase as the reactant(s).

 The number of collisions between reactants and catalyst is at a maximum

because the catalyst is uniformly dispersed throughout the reaction mixture.
Enzymes
Enzymes, catalysts that occur naturally in living organisms, are almost all protein
molecules with typical molecular masses of 20,000–100,000 amu

 Some are homogeneous catalysts that react in aqueous solution within a cellular
compartment of an organism.
 Others are heterogeneous catalysts embedded within the membranes that
separate cells and cellular compartments from their surroundings.
 The reactant in an enzyme-catalyzed reaction is called a substrate
 Enzymes…

 The enzyme glucosidase converts the sugar maltose into two glucose sugars. Active
site residues in red, maltose substrate in black, and NAD cofactor in yellow.
 Enzymes…
Coenzymes

 Organic molecules that assist enzymes and facilitate catalysis are co-factors called
coenzymes.

 If the coenzyme is very tightly or covalently bound to the enzyme, it is referred to as

a prosthetic group.

 Enzymes without their co-factors are inactive and referred to as apoenzymes.

 Enzymes containing all of their co-factors are called holoenzymes.

Co-enzyme A structure
General mechanisms of action

 If there is a magical component to life, an argument can surely be made for it

being catalysis.
 Enzymatically catalyzed reactions are orders of magnitude faster than
uncatalyzed and chemical-catalyzed reactions. The secret of their success lies in
a fundamental difference in their mechanisms of action.
 General mechanisms of action…
Changes
 In the midst of the catalytic action, an enzyme is
temporarily changed. In fact, it is the ability of an
enzyme to change that leads to its incredible
efficiency.

 These changes may be subtle electronic ones,

more significant covalent modifications, or
structural changes arising from the flexibility
inherent in enzymes, but not present in chemical
catalysts.

 Flexibility allows movement and movement facilitates alteration of electronic

environments necessary for catalysis.
 Enzymes are, thus, much more efficient than rigid chemical catalysts as a result of
their abilities to facilitate the changes necessary to optimize the catalytic process.
 General mechanisms of action…
Substrate binding and active site

 Another important difference between the

mechanism of action of an enzyme and a
chemical catalyst is that an enzyme has
binding sites that not only ‘grab’ the
substrate but also place it in a position to
be electronically induced to react, either
within itself or with another substrate.

 The enzyme itself may play a role in the electron shifts or that process may occur
as a result of substrates being oriented very close to each other.

 The location on an enzyme where substrate binds (the substrate-binding site) is also
usually the site of the reaction itself, called the active site.
General mechanisms of action…
 General mechanisms of action…

Enzyme flexibility

 As large and complex molecules, enzymes are somewhat flexible and responsive to
their local environments.

 Slight changes in shape, often arising from the binding of the substrate itself, help
to optimally position substrates for reaction after they bind.

Substrate binding by
methylenetetrahydrofolate reductase
 General mechanisms of action…
Induced fit

 Not only do enzymes change substrates, but that substrates also transiently change
enzyme structure. At the end of the catalysis, the enzyme is returned to its original
state.

 Koshland’s model is in contrast to the Fischer Lock and Key model, which says
simply that an enzyme has a fixed shape that is perfectly matched for binding its
substrate(s).

 Enzyme flexibility also is important for control of enzyme activity. Enzymes

alternate between the T (tight) state, which is a lower activity state and the R
(relaxed) state, which has greater activity.
 General mechanisms of action…

Fischer’s lock and key model (left)

Vs. Koshland’s induced fit model
(right).
 General mechanisms of action…

 The Koshland Induced Fit model of catalysis postulates that enzymes are flexible
and change shape on binding substrate.

Changes in shape help to

1. Aid binding of additional substrates in reactions involving more than one substrate
and/or

2. Facilitate formation of an electronic environment in the enzyme that favors

catalysis.

 This model is in contrast to the Fischer Lock and Key Model of catalysis which
considers enzymes as having pre-formed substrate binding sites.
 General mechanisms of action…
Ordered binding

 The Koshland model is consistent with multi-substrate binding enzymes that exhibit
ordered binding of substrates.

 For these systems, binding of the first substrate induces structural changes in the
enzyme necessary for binding the second substrate.

 There is considerable experimental evidence supporting the Koshland model.

 Hexokinase, for example, is one of many enzymes known to undergo significant

structural alteration after binding of substrate.

 In this case, the two substrates are brought into very close proximity by the induced
fit and catalysis is made possible as a result.
Hexokinase induced fit
 General mechanisms of action…

Sequential ordered binding

 In pyruvate dehydrogenase, NADH must bind prior to the binding of pyruvate.

 In this case, binding of the NADH changes the enzyme shape/environment so that
pyruvate can bind and without binding of NADH, the substrate cannot access the
pyruvate binding site.

 This type of multiple substrate reaction is called sequential ordered binding,

because the binding of substrates must occur in the right order for the reaction to
proceed.
 General mechanisms of action…

Random binding

 A second mechanism of binding/catalysis is exhibited by creatine kinase which

catalyzes the following reaction:

 For this enzyme, substrates can bind to it in any order.

 Creatine kinase displays sequential random binding. It is worth mentioning that

random binding is not inconsistent with Koshland’s induced fit model.

 Rather, random binding simply means that the enzyme’s induced fit doesn’t affect
substrate binding sites and involves other parts of the enzyme.

 In summary, sequential binding can occur as ordered binding or as random binding.

 General mechanisms of action…

Double displacement reactions

 Not all enzymes that catalyze multi-substrate

reactions, though, bind A and B by the sequential
mechanisms above.

 This other category of enzyme includes those that

exhibit what are called “ping-pong” (or double
displacement) mechanisms.

 In these enzymes, the enzyme functions as

both a catalyst and a carrier of a group between
individually bound substrates. Examples of this
type of enzyme include the class of enzymes
Double displacement
known as transaminases. reaction mechanism of a
transaminase
 General mechanisms of action…
Summary
Enzyme kinetics (Michaelis-Menten kinetics)
To understand how an enzyme enhances the rate of a reaction, we must understand
enzyme kinetics.
where E is enzyme, S is substrate, and P is
product. In this scheme, ES is the Enzyme-
Substrate complex, which is simply the
enzyme bound to its substrate.

We could define the ES state a bit further with

Where ES* is the activated state and EP is the enzyme-product complex before
release of the product.
 Velocity Enzyme kinetics …
 The first consideration we have is velocity.

Velocity = the rate of creation of product over time, measured as the

concentration of product per time.
 Enzyme kinetics …
Velocity

Equilibrium

 In a closed system (in which an enzyme operates), all reactions will advance towards
equilibrium.

 Enzymatically catalyzed reactions are no different in the end result from non-
enzymatic reactions, except that they get to equilibrium faster.

 At equilibrium, the ratio of product to reactant does not change. That is a property of
equilibrium.

 Since the system is closed, the concentration of product over time will not change.
The velocity will thus be zero under these conditions
 Enzyme kinetics …
Velocity
 In Michaelis-Menten kinetics, velocity is measured as initial velocity (V0).

 This is accomplished by measuring the rate of formation of product early in

the reaction before equilibrium is established and under these conditions,
there is very little if any of the reverse reaction occurring.

 The other two assumptions are related. First, we use conditions where there is
much more substrate than enzyme..
 Enzyme kinetics …
Waiting for substrate

 If the substrate is not in great excess, then the enzyme’s conversion of substrate to
product will occur much faster than the enzyme can bind substrate.

 Thus, the enzyme would “wait” for substrate to bind if there were not sufficient
amounts of it to bind to the enzyme in a timely fashion (when substrate
concentration is low).

 This would not give an accurate measure of velocity, since the enzyme would
be inactive a good deal of the time. Because of this, we assume saturation of
the enzyme with substrate will give a maximal velocity of the reaction.
 Enzyme kinetics …
Steady state

Concentration of product (P), substrate (S), enzyme (E), and enzyme-substrate

complex (ES) versus time for an enzymatic reaction
Enzyme kinetics …
 Enzyme kinetics …

Steady state versus non-steady state conditions

 Enzyme kinetics …

 Last, the high concentration of substrate combined with measuring initial conditions
results in studying reactions that are under so called steady state conditions

 When steady state occurs, the concentration of the ES complex over time is
not significantly changing during the period of analysis.

Reiterating, the three assumptions for Michaelis-Menten kinetics are

1. Measurement of initial velocity of a reaction

2. Substrate in great excess compared to enzyme
3. Reaction conditions occurring under steady state
 Enzyme kinetics …
Experimental considerations

1. Prepare 20 different tubes would be set up with enzyme buffer (to keep the enzyme
stable), the same amount of enzyme, and then a different amount of substrate in
each tube, ranging from tiny amounts in the first tubes to very large amounts in the
last tubes.
2. The reaction would be allowed to proceed for a fixed, short amount of time and then
the reaction would be stopped and the amount of product contained in each tube
would be determined.

 The initial velocity (V0) of the reaction then would be the concentration of
product found in each tube divided by the time that the reaction was allowed to
run.

3. Data from the experiment would be plotted on a graph using initial velocity (V0) on
the Y-axis and the concentration of substrate on the X-axis, each tube, of course
having a unique reaction velocity corresponding to a unique substrate
concentration.
 Enzyme kinetics …

Experimental considerations

 For an enzyme following Michaelis-

Menten kinetics, a curve like that shown
in the next slide would result.

Kinetics of an enzyme obeying

Michaelis-Menten kinetics
 Enzyme kinetics …
Experimental considerations

 At low concentration of
substrate, it is limiting and the
enzyme converts it into product
as soon as it can bind it.
Consequently, at low
concentrations of substrate, the
rate of increase of [P] is almost
linear with [S]
 Enzyme kinetics …
Non-linear increase

 As the substrate concentration increases, however, the velocity of the reaction in

tubes with higher substrate concentration ceases to increase linearly and instead
begins to flatten out, indicating that as the substrate concentration gets higher and
higher, the enzyme has a harder time keeping up to convert the substrate to
product.
 Enzyme kinetics …
Saturation

When the enzyme becomes completely saturated with substrate, it will not have to
wait for substrate to diffuse to it and will therefore be operating at maximum velocity.

For an enzyme following Michaelis-Menten kinetics will have its velocity (v) at any
given substrate concentration given by the following equation:
Enzyme kinetics …
 Enzyme kinetics …
Vmax

 It refers to the maximum velocity of an enzymatic reaction.

 Maximum velocity for a reaction occurs when an enzyme is saturated with substrate.
Saturation is important because it means (per the assumption above) that none of
the enzyme molecules are “waiting” for substrate after a product is released.

 Saturation ensures that another substrate is always instantly available.

 The unit of Vmax is concentration of product per time = [P]/time.

 Enzyme kinetics …
Km

 Referred to as the Michaelis constant, Km is the substrate concentration that

causes the enzyme to work at half of maximum velocity (Vmax/2).

 Is the affinity an enzyme has for its substrate.

 The value of Km is inversely related to the affinity of the enzyme for its substrate.
 Enzymes with a high Km value will have a lower affinity for their substrate (will take
more substrate to get to Vmax/2) whereas those with a low Km will have high affinity
and take less substrate to get to Vmax/2.
 The unit of Km is concentration.
 Affinities of enzymes for substrates vary considerably, so knowing Km helps us to
understand how well an enzyme is suited to the substrate being used. Measurement
of Km depends on the measurement of Vmax.
Kcat Enzyme kinetics …
 Remember that Vmax depended on the amount of enzyme used. For this, we
use the Kcat, also known as the turnover number. Kcat is a number that requires
one to first determine Vmax for an enzyme and then divide the Vmax by the
concentration of enzyme used to determine Vmax.

Kcat = Vmax /[Enzyme]

 Since Vmax has units of concentration per time and [Enzyme] has units of
concentration, the units on Kcat are time-1.

 While that might seem unintuitive, it means that the value of Kcat is the number
of molecules of product made by each molecule of enzyme in the time given.

 So, a Kcat value of 1000/sec means each enzyme molecule in the reaction at
Vmax is producing 1000 molecules of product per second.

 Note that since Kcat is a calculated value, it cannot be read from a V vs [S]
graph as Vmax and Km can.
Enzyme kinetics …
Dissociation constant (Kd) Enzyme kinetics …
 In studying proteins and ligands, it is important to understand the “tightness” with
which a protein (P) “holds onto” a ligand (L).

 This is measured with the dissociation constant (Kd ).

 The formation of a ligand-protein complex LP occurs as

 The dissociation of the complex,

therefore, is the reverse of this
reaction, or
• Where multiple molecules bond
together, such as
 so the corresponding dissociation
constant is defined as
Derivation of Michaelis-Menten equation

Michaelis-Menten derivation for simple steady-state kinetics

 The Michaelis-Menten equation is a mathematical model that is used to analyze

simple kinetic data. The model has certain assumptions, and as long as these
assumptions are correct, it will accurately model your experimental data. The
derivation of the model will highlight these assumptions.

 In an enzyme catalyzed reaction the substrate initially forms a reversible complex

with the enzyme (i.e. the enzyme and substrate have to interact for the enzyme to
be able to perform its catalytic function). The standard expression to show this is the
following:
 Derivation of Michaelis-Menten equation…

ASSUMPTION #1:

There is no product present at the start of the kinetic analysis

Therefore, as long as we monitor initial reaction rates we can ignore the reverse
reaction of E+P going to ES

ASSUMPTION #2:

 During the reaction an equilibrium condition is established for the binding and
dissociation of the Enzyme and Substrate (Briggs-Haldane assumption). Thus,
the rate of formation of the ES complex is equal to the rate of dissociation plus
breakdown
 Derivation of Michaelis-Menten equation…
ASSUMPTION #3:

[E] << [S]

The enzyme is a catalyst, it is not destroyed and can be recycled, thus, only small
amounts are required
The amount of S bound to E at any given moment is small compared to the amount of
free S
It follows that [ES] << [S] and therefore [S] is constant during the course of the
analysis (NOTE: this assumption requires that the reaction is monitored for a short
period, so that not much S is consumed and [S] does not effectively change - see next
assumption)

ASSUMPTION #4:

Only the initial velocity of the reaction is measured

[P] = 0 (reverse E + P reaction can be ignored)
[S] » [S]initial

ASSUMPTION #5:

The enzyme is either present as free enzyme or as the ES complex

[E]total = [E] + [ES]
Michaelis-Menten derivation using above assumptions:

Rate of ES formation = k1[E][S] + k-2[E][P]

Assumption #1 says we can ignore the k-2 reaction, therefore:

Rate of ES formation = k1[E][S]

Assumption #5 says [E] = [E]total - [ES], therefore:

Rate of ES formation = k1([E]total - [ES])[S]

The rate of ES breakdown is a combination of the dissociation and the conversion to

product:

Rate of ES breakdown = k-1[ES] + k2[ES]

Rate of ES breakdown = (k-1 + k2)[ES]

Assumption #2 says the rate of ES formation equals the rate of breakdown:

k1([E]total - [ES])[S] = (k-1 + k2)[ES]

 Derivation of Michaelis-Menten equation…
Rearrange to define in terms of rate constants:

([E]total - [ES])[S] / [ES] = (k-1 + k2) / k1

([E]total [S] / [ES]) - [S] = (k-1 + k2) / k1, Define a new constant, Km = (k-1 + k2) / k1

([E]total [S] / [ES]) - [S] = Km

Solve for the [ES] term (for reasons that will be given in the next step):

[ES] = [E]total [S] / (Km + [S])

The actual reaction velocity measured at any given moment is given by:

V = k2[ES]
 Derivation of Michaelis-Menten equation…
Multiple both sides of the above equation by k2:

k2[ES] = k2 [E]total [S] / (Km + [S])

thus

V = k2 [E]total [S] / (Km + [S])

The maximum possible velocity (Vmax) occurs when all the enzyme molecules
are bound with substrate [ES] = [E]total, thus:

Vmax = k2 [E]total

Substituting this into the prior expression gives:

V = Vmax [S] / (Km + [S])

This is the mathematical expression that is used to model your experimental kinetic
data

It is known as the Michaelis-Menten equation

 How does the active site of an enzyme work ? Explain from the electron and
electric field production perspectives

Some references are given below. You can add your own too

1. Charge Density in Enzyme Active Site as a Descriptor of Electrostatic

Preorganization

3. Research on Electric Field—Induced Catalysis Using Single—Molecule Electrical

Measurement

4. Enzyme Electric Fields

https://www.science.org/content/blog-post/enzyme-electric-fields