ECH 3201 BIOCHEMICAL

ENGINEERING

CHAPTER 8 & 9
ENZYME KINETICS
PART1
DR. SURYANI KAMARUDIN
8 NOVEMBER 2018

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Content
1. Nomenclature and units
2. Mechanism of simple catalysis
3. Michaelis-Menten kinetics
4. Allosteric enzymes
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What is Enzyme?
1. Enzyme… from the Greek ένζυμο,
énsymo,
2. which means én ("in") and simo ("yeast")
3. a biological catalyst, mostly protein
4. have complex structure (sequence of
amino acids) act only upon a specific
substrate
5. accelerate or catalyze chemical
reactions (A--->B) in cells by breaking
old covalent bonds & forming new
covalent bonds
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Introduction
1. Enzymes are very effective, specific, and
versatile biocatalysts
2. high MW protein (15,000 < MW < 106 Daltons),
glycoprotein, or RNA molecules
3. Some protein enzymes (apoenzyme) require a
nonprotein group for their activity as
a cofactor, (e.g. metal ions, Mg, Zn, Mn, Fe) or,
a coenzyme, (e.g., NAD, FAD, CoA) or,
some vitamins
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Ribonucleic acid (RNA) is a polymeric molecule essential in various biological

roles in coding, decoding, regulation and expression of genes.
RNA and DNA are nucleic acids, and, along with lipids, proteins and
carbohydrates, constitute the four major macromolecules essential for all known
forms of life.
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Nomenclature

 named by adding the suffix -ase to

1. the end of the substrate converted,
such as urease,
2. or the reaction catalyzed, such as
alcohol dehydrogenase.

Refer to: Chapter 3 - Bioprocess Engineering: Basic

Concepts, Shuler and Kargi, Prentice Hall, 2002
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Major Classes of Enzymes (Table 3.1)

 Oxidoreductases
• oxidation – reduction reactions
 Transferases
• transfer of whole functional groups (e.g. NH2 group)
 Hydrolases
• Hydrolysis reactions involving various functional
groups
 Lyases
• Additions to double bonds
 Isomerases
• Isomerization reactions
 Ligases
• formation of bonds with ATP cleavage
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The Enzyme Commission number (EC number) is a numerical

classification scheme for enzymes, based on the chemical reactions
they catalyze.
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Enzyme Unit (U)

 The enzyme unit (U) is a unit for the amount of a
particular enzyme.
 One U is defined as that amount of enzyme that
catalyzes the conversion of 1 micro mole of
substrate per minute.
 The enzyme unit was adopted by the
International Union of Biochemistry in 1964.
 katal, the unit recommended by the General
Conference on Weights and Measures in 1978
and officially adopted in 1999.
 One katal is the amount of enzyme that converts
1 mole of substrate per second.
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The Nature of Enzyme Catalysis

●Enzyme provides a catalytic surface
●This surface stabilizes transition state
●Transformed transition state to product
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How Enzymes Work (3.2) (page 58)

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Enzymes catalyze reactions by lowering

the energy of activation, Ea
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Enzyme Kinetics (3.3 pg. 60)

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ENZYME KINETICS
 single substrate enzyme catalyzed reactions kinetic model
developed by :
V. C. R. Henri in 1902 and
L. Michaelis and M. L. Menten in 1913.
 often referred to as Michaelis-Menten kinetics or saturation
kinetics (Fig.3.3).
 based on data from batch reactors with constant liquid
volume
 initial substrate, [So], and enzyme, [Eo], concentrations are
known.
 enzyme has a fixed no. of active sites to which substrates can
bind.
 at high substrate concentrations, all these sites may be
saturated.
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---- (3.1)
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Enzyme Kinetics summary

--- A

--- B

--- C
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1) The rapid equilibrium assumption

 Used by Henri, Michaelis and Menten .
 Assuming a rapid equilibrium between the
enzyme and substrate to form an [ES] complex,
 Use the equilibrium coefficient to express [ES]
in terms of [S].
 The equilibrium constant is,

Michaelis-Menten constant, Km’

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Michaelis-Menten Kinetics (eqn. 3.8)

where maximum forward velocity

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2). The quasi-steady-state

assumption.
 Proposed by G. E. Briggs and J. B. S. Haldane
in a closed system (batch reactor) :
the [So] >>>[Eo]
since [Eo] was small, d[ES]/dt =0.
 In many instances, the [Eo] <<<< [So], so this
assumption is an excellent approximation after
start-up.
 Computer simulations of the actual time course
represented by eqs. A, B & C (SLIDE 19) have shown that
in a closed system the quasi steady-state hypothesis
holds after a brief transient if [So] » [Eo] (for example,
100x).
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 Time course of the formation of an enzyme/substrate complex

(ES) and initiation of the steady state
 Data from actual experiment on enzyme
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Quasi steady-state assumption

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Km or Km’

Since Km results from the

more general derivation, we
will use it in the rest of our
discussion
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Refer page 64
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Refer page 65
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Refer page 66
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Allosteric enzymes
 Some enzymes have more than one substrate
binding site.
 The binding of one substrate to the enzyme
facilitates binding of other substrate molecules.
 This behavior is known as allostery or
cooperative binding,
 regulatory enzymes show this behavior.
 The rate expression in this case is

Where,
n = cooperativity coefficient and,
n > 1 indicates positive cooperativity.
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Figure 3.8, pg 68. Michaelis-Menten kinetics vs

allosteric enzyme kinetics, indicating a sigmoidal
shape of v - [S] plot for allosteric enzymes.
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 Content

 To classify and explain the inhibited

enzyme kinetics
 To explain the effect of pH and
temperature (T) on the enzyme
activity

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 What does Km mean?
1. Km = [S] at ½ Vmax
2. Km is a combination of rate constants describing the
formation and breakdown of the ES complex
3. Km is usually a little higher than the physiological [S]

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 Plot of rate (V) against substrate concentration ([S])

V max

Initial
rate, V
V max/
2

KM Substrate concentration, [S]

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 Vmax [S] Michaelis-Menten
v= equation
KM + [S]
Taking the reciprocal of both sides can give

1 KM 1 1 38

= . +
v Vmax [S] Vmax

This is in the general form y=mx+c

𝟏 𝟏
i.e. a plot of against will give straight line
𝐯 [𝐒]

This is a Lineweaver-Burk plot

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 Lineweaver-Burk plot

1/V

slope =
KM/ V
-1/K max
M
1/V
max
1/[S]

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 Michaelis-Menten equation

Vmax [S]
v=
KM + [S]

KM is the substrate concentration at which the reaction

proceeds at half of the maximum rate (Vmax)

KM is a useful indicator of the affinity of an enzyme for the substrate

• An enzyme with a low KM has a high affinity for the substrate

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 Vmax = velocity where all of the enzyme is bound
to substrate
(enzyme is saturated with S)

Km = [S] @ ½ Vmax
(units moles/L = M)
𝟏
(𝟐 of enzyme bound to S)

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 1. INHIBITED ENZYME KINETICS
Compounds that bind to enzymes and reduce
their activity are known to be ENZYME
INHIBITORS.

IRREVERSIBLE INHIBITORS
i.e. heavy metals (lead, cadmium, mercury)
 form a stable complex with enzyme and,
 may be reversed only by using chelating agents
such as EDTA and citrate.

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 1. INHIBITED ENZYME KINETICS
Studies on INHIBITORS are useful for:
1. Systematic studies to learn about how enzymes interact with
their substrates.
2. Role of inhibitors in enzyme regulation.
3. Drugs if they inhibit abnormal or harmful biochemical
reactions:
 penicillin, ampicillin, et al.: interfere with the synthesis of bacterial
cell walls.
 methotrexate - anti-cancer drug that affects DNA metabolism in
actively growing cells
 viagra - interfere with nitric oxide breakdown (NO is a vasodilator)
‘agent that causing widening of the blood vessels’
4. Understanding the role of biological toxins. A drug or other
compound produced by living organisms. It is often a commercially
important product of genetic modification.
 Arsenate - mimics phosphate esters in enzyme reactions, but are
easily hydrolyzed.
 Amino acid analogs - useful herbicides (i.e. roundup)
 Insecticides - chemicals
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Dr. Suryani Kamarudin
 ENZYME INHIBITORS
Molecules which slow down or prevent an enzyme reaction

Irreversible
• Bind very tightly
• Generally form covalent bonds

Reversible
• Non-covalent
i. Competitive - binds at the active site
ii. Non-competitive - binds at another site on
enzyme

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 REVERSIBLE INHIBITORS
 Reversible inhibitors may separate more
easily from the enzyme after binding.
 Substrate may act as an inhibitor in some
cases.
 The 3 major classes:
A. COMPETITIVE INHIBITOR (CI)
B. NONCOMPETITIVE INHIBITOR (NCI)
C. UNCOMPETITIVE INHIBITOR (UCI)

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 A. Competitive Inhibitors (CI)
 usually substrate analogs
 compete with substrate for the active site of the
enzyme
 Inhibitor binds to the same site on the enzyme as the
substrate. More rarely inhibitor binds to different site,
causing conformational change in active site so substrate
can't bind.
 Inhibitor only binds to the free enzyme
 Inhibitor is usually structurally very similar to the substrate.
 Succinate (an ester of succinic acid) / Malonate
 ATP (a chemical compound nucleotide in living organisms
that releases energy for cellular reactions when it converts to
ADP) / AMP (a compound nucleotide involved in energy
transfer reactions in living cells)

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 A. Competitive Inhibitors (CI)

EI COMPLEX

 The inhibitor reduces the amount of E by the formation of EI complex

 The inhibitor does not affect the ES complex after it has formed
 The dissociation constant for the inhibitor is KI = [E][I]/[EI]
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 A. Competitive Inhibitors (CI)
 The net effect of
competitive inhibition
is an increased value
of K’m, app and,
therefore, reduced
reaction rate.
 Can be overcome by
high concentrations of
substrate, increasing
[S].

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 A. Competitive Inhibitors (CI)
 There are 2 anticipated consequences of this
additional competitive equilibrium:
1. Vm is unchanged: At high levels of substrate all of the
inhibitor is displaced by substrate
2. Km is increased: Higher substrate concentrations are
required to reach the maximal velocity
 Steady-state analysis of the effect of the inhibitor
shows that Km is increased by a factor of (1+[I]/KI).
 The resulting form of the Michaelis-Menten equation
is:

Km’ app
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 A. Competitive Inhibitors (CI)

• Prevents S from binding, so increases apparent Km’

• no effect on Vm

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 A. Competitive Inhibitors (CI)
• CI increases apparent Km, but doesn't affect Vm.
• High [S] overcomes effect of I.

• Km higher with
inhibitor than without
inhibitor
• Vm not changed

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 A. Competitive Inhibitors (CI)

•CI binds free enzyme

•Competes with substrate for enzyme binding.
•Raises Km without effecting Vmax
•Can relieve inhibition with more S
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 A. Competitive Inhibitors (CI)
Competitive inhibition

Vmax
1 /V + inhibitor
+ inhibitor
V
Vmax/
2 uninhibited

KM KM, app 1/[S]

[S]

Vmax is unaffected
Km is increased

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B. Noncompetitive Inhibitors (NCI)

 No substrate analogs and bind on sites other than the
active site and reduce enzyme affinity to the substrate.

Dr. Suryani Kamarudin

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B. Noncompetitive Inhibitors (NCI)

• The net effect is a

reduction in Vm
• High [S] would not
overcome this type of
inhibition.
• Other reagents needed
to block binding of the
inhibitor to the enzyme.
• If the complex ESI can
form product
Vm is reduced and Km’ is
increased

Dr. Suryani Kamarudin

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B. Noncompetitive Inhibitors (NCI)

•NI can bind free E or ES complex

•Lowers Vmax, but Km remains the same
•NI’s don’t bind to S binding site therefore don’t effect Km
•Alters conformation of enzyme to effect Dr.
catalysis but not
Suryani Kamarudin
substrate binding
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B. Noncompetitive Inhibitors (NCI)

• Enzyme can bind both substrate and inhibitor simultaneously.

• ESI complex can't make product, so it must dissociate in order
for catalysis to occur.

Dr. Suryani Kamarudin

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B. Noncompetitive Inhibitors
Non-competitive inhibition (NCI)
V max
1 /V + inhibitor

V
Vmax /
2 uninhibited
+ inhibitor

KM 1/[S]
[S]

Vmax is decreased
KM is unaffected

Dr. Suryani Kamarudin

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C. Uncompetitive Inhibitor (UCI)

Inhibitor binds only to E-S complex.
• Binding site for I is created only upon S binding.
• There’s no detectible EI complex, only E, ES, and ESI, but ESI
can’t make product.
• High [S] does NOT overcome effect of I.

Dr. Suryani Kamarudin

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C. Uncompetitive Inhibitor (UCI)

•UI binds ES complex

•Prevents ES from proceeding to E + P or back to E
+ S.
•Lowers Km & Vmax, but ratio of Km/Vmax remains
the same
•Occurs with multisubstrate enzymes Dr. Suryani Kamarudin
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C. Uncompetitive Inhibitor (UCI)

Dr. Suryani Kamarudin

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C. Uncompetitive Inhibitor (UCI)

• Inhibitor binds only to E-S complex.
• Binding site for I is created only upon S binding.
• There’s no EI complex, only E, ES, and ESI, but ESI can’t make
product.

Dr. Suryani Kamarudin

 Types of Reversible Enzyme Inhibitors

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 EFFECT OF PH AND TEMPERATURE
 the rate of an enzyme-catalyzed reaction
increases as the temperature is raised.
 A ten degree Centigrade rise in
temperature will increase the activity of
most enzymes by 50 to 100%.
 Variations in reaction temperature as
small as 1 or 2 degrees may introduce
changes of 10 to 20% in the results.
 many enzymes are adversely affected by
high temperatures.
 As shown in Figure 13, the reaction
rate increases with temperature to a
maximum level, then declines with
further increase of temperature.
 most animal enzymes rapidly become
denatured at temperatures above 40oC,
enzyme determinations are carried out
below that temperature.
 Storage of enzymes at 5oC or below is
generally the most suitable.
 Some enzymes lose their activity when
frozen.

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 EFFECT OF PH AND TEMPERATURE
 Enzymes are affected by changes in pH.
 The most favorable pH value - the point where the enzyme is most
active - is known as the optimum pH.
 This is graphically illustrated in Figure 14.
 Extremely high or low pH values generally result in complete loss of
activity for most enzymes.
 pH is also a factor in the stability of enzymes. As with activity, for
each enzyme there is also a region of pH optimal stability.
 The optimum pH value will vary greatly from one enzyme to another.

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 THANK YOU

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