CHAPTER 6

Basic Concepts of Enzyme Action

1
 Chapter 6 Outline
6.1 Enzymes Are Powerful and Highly Specific Catalysts

6.2 Many Enzymes Require Cofactors for Activity

6.3 Free Energy Is a Useful Thermodynamic Function for

Understanding Enzymes

6.4 Enzymes Facilitate the Formation of the Transition

State

2
A Comparison of Rate Enhancements by Selected Enzymes.
K2 Ξ kcat  conversion of ES to product per unit time.

Leventhal’s paradox is not the only paradox that confronts us.

3
Many Enzymes Require Cofactors for Activity.

Cofactors are small molecules that some enzymes require for activity. The
two main classes of cofactors are coenzymes—organic molecules derived
from vitamins—and metals.

Tightly bound coenzymes are called prosthetic groups.

An enzyme with its cofactor is referred to as the ‘holoenzyme’. Without the

cofactor, the enzyme is referred to as an ‘apoenzyme’.

4
Enzyme Cofactors

5
ΔG0’ and Keq are opposite sides of the same coin. Will a reaction proceed as
written?
Kc = [P]eq/[Q]eq Kc= [P]eq/[R]eq > 1.0
Kc = [P]eq/[R]eq = 1.0
Kc = [P]eq/[R]eq < 1.0

6
1. A reaction will occur without the input of energy or spontaneously only if ΔG is negative. Such
reactions are called exergonic reactions. Many of the reactions that we encounter in Biochemistry
are exergonic.

2. A reaction will not occur if the ΔG is positive. These reactions are called endergonic reactions.
If a reaction is at equilibrium, there is no net change in the amount of reactant or product. At
equilibrium, ΔG = 0.

3. The ΔG of a reaction depends only on the free energy difference between reactants and
products
and is independent of how the reaction occurs.

4. The ΔG of a reaction provides no information about the rate of the reaction.

7
ΔGo symbolizes the standard free energy change at pH 7.

At equilibrium, ΔG = 0, so for the reaction in question:

[P]

[R]

or
[P]

[R]
8
The equilibrium constant for the reaction under standard conditions is:

[A] = 1M
[B] = 1M
Thus, [C] = 1M
[D] = 1M

which can be arranged to give:

or
Be careful here!

9
The more exergonic a reaction is, the larger the equilibrium constant will be.
The more endergonic a reaction is, the smaller the equilibrium constant will
be.

Note that the ΔG of the reaction can be larger than, smaller than or equal to
ΔGo, depending on the concentrations of the reactants and products.

ΔG0’ = -RT ln[P]eq/[R]eq = - # (Keq > 1.0) Reactants

ΔG0’ = -RT ln[P]eq/[R]eq = + # (Keq < 1.0) G

Products

ΔG0’ = -RT ln[P]eq/[R]eq = 0 (keq = 1.0)

ΔG0’ = Gproducts – G reactants

10
 Vmax

The Rectangular Hyperbola – describes a saturation process,

i.e., approaching a maximum value V max for an enzyme OR
Bmax (maximum binding load) for transport of a ligand, e.g., O2
Velocity (product/unit time)

by a protein such as Myoglobin. Note that the uncatalyzed

reaction attains the same V max but it takes hours-years vs.
seconds in the presence of the enzyme.

Very slow and ‘linear’

11
 A chemical reaction proceeds through a transition state, i.e., a state
between starting substrate and product-a molecular form that is no
longer substrate but not yet product.

The transition state is designated by the double dagger.

The transition state (not the substrate) is the most
complementary, i.e., ‘best fit’ to the active site.

The energy required to form the transition state from the

substrate is called the activation energy, symbolized by

The sole purpose of the enzyme is to facilitate the formation of the transition state.

12
 Our fundamental thermodynamic concept

The larger ΔGǂ, the

smaller the number of
molecules that have
sufficient thermal energy
to achieve, i.e., to get over
the energy barrier, the
transition state free
energy.

ΔΔGcat =ΔG(uncat) – ΔG(cat)

13
 Catalysis and Kinetics
Kinetics is the study of reactions speeds-the rate at which a reaction proceeds, and the factors that
can affect this rate. Catalysts are one such factor. Temperature is another, affecting the speed of
the reactive particles and therefore the rate at which they collide.

Collision Theory-The simplest and most descriptive model of how reactions occur is known as
collision theory. According to this model the atoms, and/or molecules, that constitute the reactants
are like billiard balls, zooming around a table. For a reaction to occur they need to collide, and to
do so with enough force to overcome the activation energy barrier. The moving particles have
kinetic energy; if they have enough kinetic energy they can break chemical bonds and transfer their
energy into new chemical bonds (which store it as chemical energy). As well as having sufficient
energy, one reactive particle may also need to hit the other at just the right spot-each particle has a
reactive site, and this is where the collision must occur.

Turning up the heat-The temperature of a substance or mixture is a measure of the average

kinetic energy of the particles making it up. Adding heat to a substance increase its temperature
because the heat energy is converted into kinetic energy and the average kinetic energy of the
particles goes up. In other words, a higher temperature means that particles have more energy, on
average. Particles that have more energy are moving around more and are thus more likely to
collide. Another option for boosting reaction rates is to increase the concentration of the reactants.
Essentially, having more reactive particles in a given volume of space increases the chances that
they will collide, and the more collisions there are the more likely it is that a reaction will occur. This
is extremely important because cells operate at a constant temperature.

14
 Enzymes and Substrates – A ‘contact’ sport

Enzymes bring substrates (the molecule being acted upon) together to form an
enzyme-substrate (ES) complex in a particular region of the enzyme called the
active site in order to convert the substrate to products.

The interaction of the enzyme and substrates at the active site promotes the
formation of the transition state.

15
1. The active site is a three-dimensional cleft or crevice created by
amino acids, i.e., the ‘R’ groups of the primary structure.

2. The active site constitutes a small portion of the enzyme volume.

Some enzymes interact with their substrates in a ‘lock and key’
manner-most do not. Rather, the enzyme changes shape upon
substrate binding, a phenomenon called induced fit.

3. Active sites create unique microenvironments, e.g., pKa values can

be altered (we have seen this before, e.g., the picking up of H+s by
Hb β chains.

4. The interaction of the enzyme and substrate at the active site

involves multiple weak interactions (Why do you not want strong
permanent bonds made?).

5. Enzyme specificity depends on the molecular architecture at the

active site.
16
Active Site of Lysozyme

Residues (amino acids) far apart from

one another in the primary (10)
structure (coded for by the DNA
molecule) are close to one another in
the folded tertiary (30) structure.

A B CD E F

17
‘Lock and Key’ Model of substrate binding.

18
‘Induced Fit’ Model of substrate binding

19
Binding energy is the free energy released upon interaction of the enzyme and
substrate.

Binding energy is greatest when the enzyme interacts with the transition state,
thus facilitating the formation of the transition state.

Is more binding energy released when an enzyme encounters its substrate or when an
antibody encounters it eliciting antigen? What is the implication here?

20