CHAPTER 2

ENZYME KINETICS &

INDUSTRIAL
ENZYMOLOGY

In this topic, you will learn about:

1) Nature of enzyme action.
2) Michaelis-Menten Equation,
Lineweaver-Burk, & other plots.
3) Steady state approach.
4) Enzyme inhibition; competitive &
non-competitive inhibition.
5) Temp & pH effect.

1) NATURE OF
ENZYME ACTION

DEFINITION of ENZYMES
Biological catalysts that are protein
molecules in nature.
Produced by living cells (animal,
plant & microorganism).
Absolutely essential as catalysts in
biochemical reactions.

FUNCTION of ENZYMES
To catalyze the making and breaking
of chemical bonds.
To increase the rate of reaction
without themselves undergoing
permanent chemical changes.

ENZYME REACTIONS

ENERGY OF ACTIVATION

ENERGY OF ACTIVATION

In a chemical reaction, the reactants must

absorb a certain amount of energy from the
environment before a reaction can take place.
The specific amount of energy required for the
reaction to proceed is called the ENERGY OF
ACTIVATION.

ENERGY OF ACTIVATION
In reactions occurring outside of living
organisms, simply adding heat can
provide activation energy.
Inside of living organisms, however,
another method must be used. Heat is
very harmful to the cells and proteins of
plants and animals.

ENERGY OF ACTIVATION
To overcome the activation energy required for
certain reactions to take place, living organisms
employ enzymes. Enzymes function by being
able to LOWER the activation energy needed in
specific reactions.
With a lower energy requirement, more
molecules will be able to react with each other
and the reaction can swiftly occur at
temperatures able to support life.

ENERGY OF ACTIVATION

ENZYME REACTIONS
vs
CHEMICAL REACTIONS
Highly specific - catalyze only one or small
number of chemical reactions
Their rate of reaction is usually much faster
than non-biological catalyst
The reaction conditions ( T, P, pH, and so on)
are very mild.
Enzymes are comparatively sensitive or
unstable molecules and require care in their
use.

NOMENCLATURE of
ENZYMES
*Non-descriptive name such as:
curding of milk to start cheese-making
rennin
processer

pepsin hydrolyzes proteins at acidic pH

trypsin hydrolyzes proteins at mild alkaline pH

NOMENCLATURE of
ENZYMES
*Name of substrate + ase :
Substrate

Enzyme

Product

Starch

-amylase

lactose
fat
maltose

lactase
lipase
maltase

glucose + maltose +
oligosaccharides
glucose + galactose
fatty acids + glycerol
glucose

urea + H2O

urease

2NH3 + CO2

cellobiose

cellobiase

glucose

NOMENCLATURE of
ENZYMES
*Reaction which is catalyzed + ase :
Enzymes

alcohol dehydrogenase
glucose isomerase

Reaction
ethanol + NAD+
acetaldehyde + NADH2
glucose fructose

glucose oxidase

D-glucose + O2 + H2O
gluconic acid

lactic acid dehydrogenase

lactic acid pyruvic acid

COMMERCIAL APPLICATION
of ENZYMES
Industrial
enzymes

amylase, protease, glucose isomerase,

lipase, catalase, penicilin acylases
glucose oxidase, galactose oxidase,
Analytical
alcohol dehydrogenase, hexokinase,
Enzymes
muramidase, cholesterol oxidase
asparaginase, proteases, lipases,
Medical Enzymes
streptokinase
* what is HFCS ( high-fructose corn syrup)? what kind of
enzymes contribute in the process of HFCS?

ENZYME KINETICS

Study of the rates of enzyme-catalyzed

reactions
Provides information on enzyme
specificities and mechanisms

KINETICS?????

ENZYME ACTION
LOCK AND KEY MODEL
An enzyme binds a substrate in a region called
the active site.
Only certain substrates can fit the active site.
Amino acid R groups in the active site help
substrate bind.
Enzyme-substrate complex forms.
Substrate reacts to form product.
Product is released.

ENZYME ACTION
LOCK AND KEY MODEL

ENZYME ACTION
LOCK AND KEY MODEL

ENZYME ACTION
INDUCED FIT MODEL
Enzyme structure flexible, not rigid.
Enzyme and flexible active site adjust shape to bind
substrate.
This sudden change in shape can lead to the breaking of
bonds within a single substrate molecule, forming two
new molecules. Conversely, it can also bring twosubstrate molecules close enough together for them to
bond with each other, forming one new molecule.
Increases range of substrate specificity.
Shape changes also improve catalysis during reaction.

ENZYME ACTION
INDUCED FIT MODEL

ENZYME ACTION
INDUCED FIT MODEL

FORMULA FOR A SIMPLE

ENZYME-CATALYZED
REACTION

FORMULA FOR A SIMPLE

ENZYME-CATALYZED
REACTION

E = free enzyme
S = substrate
ES = enzyme-substrate complex
P = product

2) MICHAELISMENTEN EQUATION,
LINEWEAVER BURK &
OTHER PLOTS

MICHAELIS-MENTEN
EQUATION
Leonor Michaelis (1913): noticed that at constant
[enzyme] the rate of a reaction increases with
increasing [S] until a maximal velocity (Vmax) is
achieved
This saturation effect is an important distinction
versus uncatalyzed reactions
Interpretation of data: ES complexes formed until
substrate saturation occurs at which point no more
substrate binding sites (i.e., enzyme molecules) are
available

MICHAELIS-MENTEN
EQUATION

Michaelis and Menten (1913):

It is assumed that the product-releasing step (eqn
above with k2) is much slower than the reversible
reaction (k1 and k-1).
The slow step determines the rate, while the other is at
equilibrium.

MICHAELIS-MENTEN
EQUATION

max S

K m S

MICHAELIS-MENTEN
EQUATION

MICHAELIS-MENTEN
EQUATION

SIGNIFICANCE OF MICHAELISMENTEN EQUATION

Km has a unit of concentration. It is a constant for every enzyme
under specific conditions
When [S] <<< Km, Vo is directly proportional to [S]
When [S] >>> Km, Vo = Vmax
When [S] = Km, Vo = Vmax
The Km value of an enzyme indicates the concentration of the
substrate required for significant catalysis
Since Km = (k2+k-1)/k1, when k-1 >>> k2, Km = k-1/k1
Dissociation constant of ES, KES = [E][S] / [ES] = k-1/k1
When k-1 >>> k2, Km = KES. High Km = high dissociation =
weak binding between E and S
When [E]T = [ES], Vmax = k2[E]T.
k2 is called the turnover number: the rate when enzyme is
saturated with substrate

LINEWEAVER-BURK PLOT

Also called the double-reciprocal plot

1/Vo = (Km/Vmax).(1/[S]) + 1/Vmax
This is equation for a straight line y = mx + c
Slope = Km/Vmax
Y-intercept = 1/Vmax
X-intercept = -1/Km
Useful for experimental determination of Km
and Vmax

LINEWEAVER-BURK PLOT

LINEWEAVER-BURK PLOT

Km values for various

enzyme

3) STEADY STATE
APPROACH

ENZYME ACTIVITY
Enzymes are required in minute quantities and
enhance reaction rates by 1010 to 1020fold
When the enzyme is part of a crude preparation,
its concentration is in terms of units
Enzyme activity is the ability of an enzyme to
modify a reactant. 1 unit (U) is the enzyme
activity that converts 1 mole of reactant per
min under standard conditions.
The specific activity of an enzyme is defined as
the activity per unit of mass or U/mg protein

4) ENZYME INHIBITION:
COMPETITIVE & NONCOMPETITIVE
INHIBITION

ENZYME INHIBITION
Inhibitors

Cause a loss of catalytic activity.

Change the protein structure of an enzyme.
May be a reversible or irreversible inhibition.
Specific enzyme inhibitors regulate enzyme
activity and help us understand mechanism
of enzyme action. (Denaturing agents are not
inhibitors).

ENZYME INHIBITION
Inhibitors
Irreversible inhibitors form covalent or
very tight permanent bonds with the active
site of the enzyme and incapacitating the
enzyme. 3 classes: group-specific reagents,
substrate analogs, suicide inhibitors.

ENZYME INHIBITION
Inhibitors
Reversible inhibitors form an EI complex
that can be dissociated back to enzyme and
free inhibitor. 3 groups based on their
mechanism of action:
competitive, non-competitive and
uncompetitive.

COMPETITIVE INHIBITION
A competitive inhibitor
Has a structure similar to substrate
Occupies active site
Competes with substrate for binding to enzyme (active
site)
E + S = ES or E + I = EI . Both S and I cannot bind
enzyme at the same time
In presence of I, the equilibrium of E + S = ES is shifted
to the left causing dissociation of ES.
This can be reversed / corrected by increasing [S]
Vmax is not changed, Km is increased

NON-COMPETITIVE
INHIBITION
A noncompetitive inhibitor
Inhibitor binding site is distinct from substrate binding
site, it does not have a structure like substrate
Binds to the enzyme but not active site
Changes the shape of enzyme and active site
Substrate cannot fit altered active site
Can bind to free enzyme E and to ES
E + I = EI, ES + I = ESI or EI + S = ESI

NON-COMPETITIVE
INHIBITION
A noncompetitive inhibitor

Both EI and ESI are enzymatically inactive

The effective functional [E] (and [S]) is reduced
Reaction of unaffected ES proceeds normally
Inhibition cannot be reversed by increasing [S]
Km is not changed, Vmax is decreased

5) TEMPERATURE &
pH EFFECT

FACTOR AFFECTING
REACTION RATE
The study of enzyme reaction rates is called
enzyme kinetics. Enzyme kinetics are affected by:
Temperature and pH: Each enzymatic reaction
has an optimum pH and optimum temperature.
Extreme temperature or pH disrupts enzyme
structure and therefore reaction rate
Substrate concentration: The reaction rate = k [P]
/ [S]. The rate can be increased by adding more
substrate, or by removing product as it is formed

FACTOR AFFECTING
ENZYME ACTION
TEMPERATURE
Little activity at low temperature
Rate increases with temperature
Most active at optimum temperatures
(usually 37C in humans)
Activity lost with denaturation at high
temperatures

FACTOR AFFECTING
ENZYME ACTION
TEMPERATURE

FACTOR AFFECTING
ENZYME ACTION
SUBSTRATE
CONCENTRATION
Increasing substrate concentration increases
the rate of reaction (enzyme concentration is
constant).
Maximum activity reached when all of enzyme
combines with substrate

FACTOR AFFECTING
ENZYME ACTION
SUBSTRATE CONCENTRATION

FACTOR AFFECTING
ENZYME ACTION
pH

Maximum activity at optimum pH

R groups of amino acids have proper charge
Narrow range of activity
Most lose activity in low or high pH

FACTOR AFFECTING
ENZYME ACTION
pH

END OF CHAPTER 2