1. Enzymes are complex proteins that act as catalysts to speed up biochemical reactions in living cells. They do this by lowering the activation energy needed for reactions to occur. 2. The Michaelis-Menten kinetic model describes enzyme kinetics. It states that the enzyme first binds to the substrate to form an enzyme-substrate complex, and this complex then breaks down to release products and free up the enzyme. 3. The Michaelis-Menten equation relates the initial reaction rate to substrate concentration. It produces a hyperbolic curve that describes the typical saturation kinetics seen with many enzymes.

1. Enzyme
Course: B.Sc.(Biochem) II
Subject: Biomolecules I
Unit 4

2. Enzymes
What are enzymes?
 Enzymes are complex chemicals that control reactions in living cells. They are
biochemical catalysts speeding up reactions that would otherwise happen too slowly.
 The chemical which an enzyme works on is called its substrate.
 An enzyme combines with its substrate to form a short-lived enzyme/substrate complex.
Once a reaction has occurred, the complex breaks up into products and enzyme.
E + S ES EP E + P
 The enzyme remains unchanged at the end of reaction and is free to interact again with
more substrate.
How the confectioners make the runny yolk-like
inside to chocolate eggs?
The answer is : use of an enzyme

3. The role of enzymes in an organism
 Many of the complex chemicals that living organisms need cannot be made in a single
reaction. Instead a series of simpler reactions occur, one after another, forming a metabolic
pathway. A single pathway may have many steps in which each chemical is converted to
the next. A specific enzyme controls each reaction.
 Enzymes control cell metabolism by regulating how and when reactions occur. Using
this very simple pathway as an example:
A B C D
The final product is substance D, the chemical needed by the living organism. The
pathway needs three different enzymes and when D is no longer needed or if too much has
been produced, one of the three enzymes is ‘switched off’.
1

4. The Chemical nature of enzymes
Enzymes are globular proteins. They have a complex tertiary and quaternary structure
in which polypeptides are folded around each other to form a roughly spherical or globular
shape. The overall 3D shape of an enzyme molecule is very important: if it is altered, the
enzyme cannot bind to its substrate and so cannot function. Enzyme shape is maintained
by hydrogen bonds and ionic forces.
Enzymes have several important properties:
 Enzymes are specific: each enzyme usually catalyses only one reaction.
 Enzymes combine with their substrates to form temporary enzyme-substrate complex.
 Enzymes are not altered or used up by the reactions they catalyze, so can be used again
and again.
 Enzymes are sensitive to temperature and pH.
 Many enzymes need cofactors in order to function.
 Enzyme function may be slowed down or stopped by inhibitors.

5. Characteristics of Enzymes?
Enzymes
1)speed up chemical reactions
2)are required in minute amounts
3)are highly specific in their action
4)are affected by temperature
5)are affected by pH
6)Some catalyse reversible reactions
7)Some require co-enzymes
8)Are inhibited by inhibitors

6. (1) Enzymes Speed up chemical reactions
Activation Energy
with enzyme
Activation
Energy without
enzyme
Substrate
Products:
Energy
Time
• By lowering the activation energy needed to start
the reaction.

7. (2) Enzymes are required in minute amounts
Sucrose Glucose + Fructose
• They remain chemically unchanged after catalysing
the reactions.
• The same enzyme molecules can be reused over
again.
• Therefore, only a small amount of enzyme is required
to catalyse a large number of reactions
Sucrase
Chemically unchanged

8. Maltose Glucose + Glucose
(3) Enzymes are highly specific
• Each chemical reaction is catalysed by its own specific,
unique enzyme.
• This is due to every enzyme’s specific 3-d configuration.
• How the shape of an enzyme affects its function can be
explained by the “LOCK & KEY HYPOTHESIS”.
Maltase
Starch Maltose
Amylase

9. Enzyme Active Sites
Under biologically relevant conditions, uncatalyzed
reactions tend to be slow because most biological
molecules are quite stable in the neutral-pH, mild-
temperature, aqueous environment inside cells. Enzymes
greatly increase the rates of biological reactions by
providing a specific environment within which a
reaction can occur more rapidly. Enzyme-catalyzed
reactions take place within the confines of a pocket on
the enzyme called the active site. The reactant molecule
is referred to as the substrate. The surface of the active
site is lined with amino acid residues with substituent
groups that bind to the substrate and catalyze its
chemical transformation. Often, the active site encloses
the substrate, sequestering it from solution. The active
site of the enzyme chymotrypsin is highlighted in Fig. 6-
1.
2

10. ENZYME POWER!
 All reactants need to have a certain energy before they
will react. This is like an energy barrier that it has to
overcome before a reaction will occur. It is called the
activation energy.
 Enzymes are organic catalysts.
 All catalysts lower the energy barrier, allowing the
reactants (substrates) to react faster forming the products.
 Enzymes do not participate in the reaction.

11. Reactant
Product
Without enzyme: The activation
energy required is high.
With enzyme: The activation
energy required is lower.
ENZYMES
High
Low
Start Finish
Direction of reaction
Amountofenergystoredinthe
chemicals
Low energy
High energy

12. ENZYMES
Enzymes have a specific region where
the substrate binds and where catalysis
occurs. This is called the active site.
Enzymes are substrate-specific,
although specificity varies from enzyme
to enzyme.
When a substrate binds to an enzyme’s
active site, an enzyme-substrate
complex is formed. Space filling model of the yeast
enzyme hexokinase. Its active
site lies in the groove (arrowed)
3

13. LOCK AND KEY MODEL
The lock and key model of enzyme action, proposed earlier this century,
proposed that the substrate was simply drawn into a closely matching cleft
on the enzyme molecule.
Substrate
Enzyme
Products
Symbolic representation of the lock and key model of enzyme action.
1. A substrate is drawn into the active sites of the enzyme.
2. The substrate shape must be compatible with the enzymes active site in
order to fit and be reacted upon.
3. The enzyme modifies the substrate. In this instance the substrate is
broken down, releasing two products.
4

14. INDUCED FIT MODEL
More recent studies have
revealed that the process is much
more likely to involve an
induced fit.
The enzyme or the reactants
(substrate) change their shape
slightly.
The reactants become bound to
enzymes by weak chemical bonds.
This binding can weaken bonds
within the reactants themselves,
allowing the reaction to proceed
more readily.
The enzyme
changes shape,
forcing the substrate
molecules to
combine.
Two substrate
molecules are
drawn into the cleft
of the enzyme.
The resulting end
product is released
by the enzyme
which returns to its
normal shape, ready
to undergo more
reactions.
5

15. CHANGING THE ACTIVE SITE
 Changes to the shape of the active site will result in a loss of
function. Enzymes are sensitive to various factors such as
temperature & pH.
 When an enzyme has lost its characteristic 3D shape, it is said to
be denatured. Some enzymes can regain their shape while in
others, the changes are irreversible.

16. THE EFFECT OF TEMPERATURE ON ENZYME
ACTION
 Speeds up all reactions, but the
rate of denaturation of enzymes
also increases at higher
temperatures.
 High temperatures break the
disulphide bonds holding the
tertiary structure of the enzyme
together thus changing the
shape of the enzyme.
 This destroys the active sites &
therefore makes the enzyme
non – functional.
Too cold for
Enzyme to
work
Too hot for
Enzyme to
work
Optimum
Temperature
for enzyme

17. THE EFFECT OF TEMPERATURE ON ENZYME
ACTION
 The curve in the blue represents an enzyme isolated from an organism living in the
artic. These cold dwelling organisms are called psychrophiles.
 The curve in red represents an enzyme isolated from the digestive tract of humans.
 The curve in green represents an enzyme isolated from a thermophile bacteria
found growing in geothermal sea beds.

18. THE EFFECT OF PH ON ENZYME ACTION
 Like all proteins, enzymes are
denatured by extremes of pH
(acidity/alkalinity).
 The green curve is for pepsin
that digests proteins in the
stomach.
 The red curve represents the
activity of arginase that breaks
down arginine to ornithine &
urea in the liver.

19. THE EFFECT OF ENZYME
CONCENTRATION ON ENZYME ACTION
 Assuming that the
amount of substrate is
not limiting, an increase
in enzyme concentration
causes an increase in the
reaction rate.

20. THE EFFECT OF SUBSTRATE
CONCENTRATION ON ENZYME ACTION
 Assuming that the amount of enzyme is
constant, an increase in substrate
concentration causes a diminishing
increase in the reaction rate.
 A maximum rate is obtained at a certain
concentration of substrate when all
enzymes are occupied substrate (the
rate cannot increase any further).

21. THE EFFECT OF COFACTORS ON ENZYME ACTION
 Cofactors are substances that are
essential to the catalytic activity
of some enzymes.
 Cofactors may alter the shape of
enzymes slightly to make the
active sites functional or to
complete the reactive site.
 Enzyme cofactors include
coenzymes (organic molecules) or
activating ions (eg. Na+, K+..)
 Vitamins are often coenzymes (eg.
Vit B1, Vit B6…)
6

22. THE NATURE OF ENZYME INHIBITORS
 Enzyme inhibitors may or may not act reversibly:
 Reversible: the inhibitor is temporarily bound to the
enzyme, thereby preventing its function (used as a
mechanism to control enzyme activity).
 Irreversible: the inhibitor may bind permanently to the
enzyme causing it to be permanently deactivated.

23. THE NATURE OF ENZYME INHIBITORS
 Reversible Enzymes work in one of two ways:
 Competitive inhibitors: the inhibitor competes with the
substrate for the active site, thereby blocking it and
preventing attachment of the substrate.
 Non-competitive: the inhibitor binds to the enzyme (but
not at the active site) and alters its shape. It markedly
slows down the reaction rate by making the enzyme less
able to perform its function (allosteric inhibition).

24. The Role of the ES Complex (I)
The ES complex is the key to understanding the kinetic behavior of an enzyme. In 1913,
Leonor Michaelis and Maud Menten, developed a kinetic equation to explain the behavior
of many simple enzymes. Key to the development of their equation, is the assumption that
the enzyme first combines with its substrate to form an enzyme-substrate complex in a
relatively fast reversible step:
k1
E + S  ES
k-1
The ES complex then breaks down in a slower second step to yield the free enzyme and
the reaction product P:
k2
ES  E + P
k-2
If the slower second reaction limits the rate of the overall reaction, the overall rate must be
proportional to the concentration of the species that reacts in the second step, i.e., ES.
At any given instant in an enzyme-catalyzed reaction, the enzyme exists in two forms, the
free or uncombined form E and the combined form ES. At low [S], most of the enzyme is
in the uncombined E form. Here, the rate is proportional to [S] because the direction of the
first equation above is pushed toward formation of more ES as [S] increases. (Continued
on the next slide).

25. The Role of the ES Complex (II)
The maximum initial rate of the catalyzed reaction (Vmax) is observed when virtually all of
the enzyme is present in the ES complex and [E] is vanishingly small. Under these
conditions, the enzyme is saturated with its substrate, so that further increases in [S] have
no effect on rate. This condition exists when [S] is sufficiently high that essentially all the
free enzyme has been converted to the ES form. The saturation effect is a distinguishing
characteristic of enzymatic catalysts and is responsible for the plateau observed in Fig. 6-
11. The pattern seen in that figure is sometimes referred to as saturation kinetics.
When the enzyme is first mixed with a large excess of substrate, there is an initial period,
the pre-steady state, during which the concentration of ES builds up. This period is usually
too short to be easily observed, lasting just microseconds, and is not evident in Fig. 6-10.
The reaction quickly achieves a steady state in which [ES] remains approximately constant
over time. The measured V0 generally reflects the steady state, even though V0 is limited to
the early part of the reaction. The analysis of these initial rates is referred to as steady-state
kinetics.

26. Derivation of the MM Equation (I)
The kinetic curves expressing the relationship between V0 and [S] have the same general
shape (a rectangular hyperbola) for most enzymes, which can be expressed algebraically by
the MM equation. Michaelis and Menten derived this equation starting from their basic
hypothesis that the rate-limiting step in enzymatic reactions is the breakdown of the ES
complex to product and free enzyme. The MM equation is
V0 = Vmax[S]/(Km + [S]).
All these terms, [S], V0, Vmax, as well as the constant called the Michaelis constant, Km, can
be readily measured experimentally.
The derivation of the MM equation starts with the two basic steps of the formation and
breakdown of ES. Early in the reaction, the concentration of the product [P] is negligible,
and a simplifying assumption is made that the reaction P  S (described by k-2) can be
ignored. The overall reaction then reduces to
k1 k2
E + S  ES  E + P.
k-1

27. Derivation of the MM Equation (II)
V0 is determined by the breakdown of ES to form product, which is determined by [ES]
through the equation
V0 = k2[ES].
Because [ES] in the above equation is not easily measured experimentally, an alternative
expression for this term must be found. First, the term [Et], representing the total enzyme
concentration (the sum of free and substrate-bound enzyme) is introduced. Free or unbound
enzyme [E] can then be represented by [Et] - [ES]. Also, because [S] is ordinarily far
greater than [Et], the amount of substrate bound by the enzyme at any given time is
negligible compared with the total [S]. With these conditions in mind, the following steps
lead to an expression for V0 in terms of easily measurable parameters.

28. Derivation of the MM Equation (III)

29. Derivation of the MM Equation (IV)

30. Derivation of the MM Equation (V)
The MM equation describes the kinetic behavior of a great many enzymes, and all
enzymes that exhibit a hyperbolic dependence of V0 on [S] are said to follow Michaelis-
Menten kinetics. However the MM equation does not depend on the relatively simple two-
step reaction mechanism discussed above. Many enzymes that follow MM kinetics have
quite different mechanisms, and enzymes that catalyze reactions with six or eight
identifiable steps often exhibit the same steady-state kinetic behavior. Even though the
MM equation holds true for many enzymes, both the magnitude and the real meaning of
Vmax and Km can differ from one enzyme to another. This is an important limitation of the
steady-state approach to enzyme kinetics.

31. Validation of the MM Equation
The MM equation can be shown to correctly explain the V0 vs [S] curves of many enzymes
by considering limiting situations where [S] is very high or very low (Fig. 6-12). At low
[S], Km >> [S] and the [S] term in the denominator of the MM equation becomes
insignificant. The equation simplifies to V0 = Vmax[S]/Km and V0 exhibits a linear
dependence on [S], as is observed at the left side of V0 vs [S] graphs. At high [S], where [S]
>> Km, the Km term in the denominator of the MM equation becomes insignificant and the
equation simplifies to V0 = Vmax. This is consistent with the plateau in V0 observed at high
[S] in kinetic graphs.
An important numerical relationship emerges from the MM equation in the special case
when V0 is exactly one-half Vmax. Here
Vmax/2 = Vmax[S]/(Km + [S]).
On dividing by Vmax, the equation is
1/2 = [S]/(Km + [S]).
After solving for Km, we get
Km + [S] = 2[S], or Km = [S].
This is a very useful, practical definition of Km.
Km is equivalent to the substrate concentration at
which V0 is one-half Vmax.

32. Double-reciprocal Plots
Because the plot of V0 vs [S] for an enzyme-catalyzed reaction asymptotically approaches the
value of Vmax at high [S], it is difficult to accurately determine Vmax (and thereby, Km) from
such graphs. The problem is readily solved by converting the Michaelis-Menten kinetic
equation to the so-called double-reciprocal equation (Lineweaver-Burk equation) which
describes a linear plot from which Vmax and Km can be easily obtained (Box 6-1, Fig. 1). The
Lineweaver-Burk equation is derived by first taking the reciprocal of both sides of the
Michaelis-Menten equation
1/V0 = (Km + [S])/Vmax[S]
Separating the components of the numerator on the
right side of the equation gives
1/V0 = Km/Vmax[S] + [S]/Vmax[S]
Which simplifies to
1/V0 = Km/Vmax[S] + 1/Vmax.
The plot of 1/V0 vs 1/[S] gives a straight line, the y-
intercept of which is 1/Vmax and the x-intercept of
which is -1/Km.

33. The Meaning of the Km
The Km can vary greatly from enzyme to enzyme, and even for different substrates of the
same enzyme (Table 6-6). The Km is sometimes used (often inappropriately) as an
indicator of the affinity of an enzyme for its substrate. The actual meaning of the Km
depends on specific aspects of the reaction mechanism such as the number and relative
rates of the individual steps. For example, for a reaction with two steps, Km = (k2 + k-1)/k1.
If k2 is actually rate-limiting, then k2 << k-1, and Km reduces to k-1/k1, which is the
dissociation constant, Kd of the ES complex. Where these conditions hold, Km does
represent a measure of the affinity of the enzyme for its substrate. However this scenario
often doesn’t apply for an enzyme due to variances in the contributions of individual rate
constants to the overall reaction rate. Thus Km cannot always be considered a simple
measure of the affinity of an enzyme for its substrate.

34. Information Derived from Vmax
The meaning of the quantity Vmax also varies greatly from one enzyme to the next. If an
enzyme reacts via the two-step MM mechanism, then Vmax = k2[Et]. However, the number of
reaction steps and the identity of the rate-limiting step can vary from enzyme to enzyme.
Therefore, it is useful to define a more general rate constant, kcat, to describe the rate constant
of the rate limiting step(s) of any enzyme-catalyzed reaction at saturation. With the
modification that Vmax = kcat[Et], the MM equation becomes
V0 = kcat[Et][S]/(Km + [S]).
The constant kcat is a first-order rate constant and hence has the units of reciprocal time (s-1). It
is also called the turnover number for the enzyme-catalyzed reaction. It is equivalent to the
number of substrate molecules converted to product in a given unit of time on a single enzyme
molecule when the enzyme is saturated with substrate. The turnover numbers of several
enzymes are listed in Table 6-7.

35. The Specificity Constant (kcat/Km)
Together, the parameters kcat and Km can be used to evaluate the catalytic efficiency of an
enzyme. The best way to determine the catalytic efficiency of an enzyme is to determine the
ratio of kcat/Km for its reaction. This parameter, sometimes called the specificity constant, is
the rate constant for the conversion of E + S to E + P. When [S] << Km, the previous MM
equation form converts to
V0 = (kcat/Km)([Et][S]).
V0 in this case depends on the concentration of two reactants, [Et] and [S]. Therefore, this is a
second-order rate equation and the constant kcat/Km is a second-order rate constant with the
units M-1s-1. There is an upper limit to kcat/Km, imposed by the rate at which E and S can
diffuse together in an aqueous solution. This diffusion-controlled limit is 108 to 109 M-1s-1, and
many enzymes have a kcat/Km near this range (Table 6-8). Such enzymes are said to have
achieved catalytic perfection.

36. REFERENCES:
Image 1: http://postimg.org/image/hzxi8jlzz/
Image 2: http://postimg.org/image/97gjkv0v3/
Image 3: http://postimg.org/image/cfpmbncin/
Image 4 & 5: Harper’s Illustrated Biochemistry by Robert,
Murray, Hranner, Mayes and Rodwell
Image 6: http://postimg.org/image/k5rt31pgf/
Books:
1. Biochemistry, U. Satyanarayan, Elsevier India publication
2. Principles of Biochemistry by Lehninger