05.

Enzymes By the International Union of Biochemistry and

Molecular Biology (IUBMB)
Outline According to six categories
 Overview [EC #.#.#.#] class.subclass.sub-subclass.#
 Review
o Kinetic and Thermodynamic Aspects of Reactions Classification of Enzymes
o Mathematical Description of (Enzyme) Kinetics 1. Oxidoreductases
 Enzyme-Substrate Interaction Redox reactions (usually dehydrogenases)
 Michaelis-Menten Approach to Enzyme Kinetics E.g.: lactate dehydrogenase [EC 1.1.1.27]
 Inhibitors 2. Transferases
Transfer of a chemical moiety
Nature of Enzymes E.g.: kinases
E.g.: alanine transaminase [EC 2.6.1.2]
Enzymes as Effective Biological Catalysts 3. Hydrolases
 Catalysis – possible the most important function of proteins; Hydrolysis reactions
most prominent function of proteins E.g.: chymotrypsin
 Virtually no reaction in the cell occurs without enzymes E.g.: pyrophosphatase [EC 3.6.1.1]
 Almost all enzymes are PROTEINS 4. Lyases
o Exception: ribozymes (RNA) Breakage of C=C bonds
 Non-enzymatic catalysts enhance rates of reaction by a factor E.g.: aldolases (e.g. in glycolytic pathway)
of 102 to 104 E.g.: pyruvate decarboxylate [EC 4.1.1.1]
 Enzymes enhances rates of reaction by a factor of up to 1020 5. Isomerases
Transfer of functional groups w/in a molecule
 Enzymes exhibit specificity
E.g.: Aconitase (in the citric acid cycle)
o Stereoisomers
E.g.: alanine racemase [EC 5.1.1.1]
o Class of compounds
6. Ligases
 Enzymes can be fine-tuned
Joins two groups together
o They can either be speeded up or slow down depending
E.g.: synthetases
on the metabolic needs of the cell
E.g.: glutamine synthetase [EC 6.3.1.2]

Review of Kinetics and Thermodynamics

Kinetic vs. Thermodynamic Aspects in Chemical Reactions

 For the generic reaction
o aA + bB  products
 Rate of reaction
o Rate = k[A]x[B]y
o k = Ae-Ea/RT
 Equilibrium
 Enzymes are affected by o Keq = [C]c[D]d/[A]a[B]b
o Temperature – increased in temperature will increase the  Enzymes increases rates of reactions by providing an
kinetic energy of the molecule alternative mechanism where Ea is lower
 Since proteins are  Enzymes DO NOT affect ∆G° reaction
peptide chains, all
the strings bundled Enzyme Catalysis
up together For a reaction taking place at a constant temperature and
increases in kinetic pressure, e.g. in the body
energy, at some
point the IMF that
holds them together The change in free energy is
can be overcome
and denature the The change in the free energy is related to the equilibrium
protein constant, Keq, for the reaction by
o pH – some of the side chains in the polypeptide chain
could be carboxylate or ammonium which can either be
protonated or deprotonated depending on the pH and
will disrupt the IMF that holds them together For the reaction A+BP

Nomenclature of Enzymes
Common name:
Usually after the substrate or the reaction
+ase (e.g. sucrase, DNA polymerase, etc.)
Historical name (e.g. trypsin, pepsin, etc.)
Systematic name:
The Mathematical Treatment of Enzyme Kinetics Formation of Product
 Rate of reaction is also known as the velocity of reaction, also
known as the speed of reaction
 Rate = Velocity = V
 First-order reaction
o The rate of reaction is directly proportional to
concentration of reactant
 Second-order reaction
o The rate of reaction is proportional to the concentration
of reactant
 Zero-order reaction
o The rate of reaction is independent of the concentration
of reactant
Examples of Enzyme-Catalyzed Reactions
Substrate Interaction – The Nature of Enzymes Themselves Chymotrypsin catalyzes the selective hydrolysis of peptide
bonds where the carboxyl is contributed by Phe and Tyr
Enzyme-Substrate Interaction
In an enzyme-catalyzed reaction
Substrate, S: a reactant
Active Site: the small portion of the enzyme surface
where the substrate(s) becomes bound by noncovalent forces
e.g. hydrogen bonding, electrostatic attractions, van der
Waals attractions

Chymotrypsin also catalyzes hydrolysis of the ester bond of p-

nitrophenyl esters
Two models have been developed to describe the formation of
the enzyme-substrate complex
Lock-and-key-model: substrate binds to that portion of
the enzyme with a complementary shape
Induced fit model: binding of the substrate induces a
change in the conformation of the enzyme that results in a
complementary fit
Dependence of reaction velocity, V, on p-nitrophenyl acetate
concentration, [S] in a reaction catalyzed by chymotrypsin.

Enzyme Catalysis
Glucose-Hexokinase Complex
Aspartate transcarbamylase (ATCase) catalyzes this reaction

Follow rate of reaction when [carabomyl phosphate] is constant

Energy profile for formation of an E-S complex
Comparison of the shapes of the curves for chymotrypsin-
catalyzed reaction and for aspartate transcarbamoylase- Plotting initial rate, Vinit, versus [S]
catalyzed reaction

Comparison of different portions of the Michaelis-Menten Plot

The Michaelis-Menten Approach to Enzyme Kinetics

Michaelis-Menten Model
For an enzyme-catalyzed reaction

The rates of formation and breakdown of ES are given

by these equations

When [S] = KM, the equation reduces to

At the steady state (concentration of ES is constant)

When a steady state is reached, the concentration of

free enzyme is the total less that bound in ES

Substituting for the concentration of free enzyme and

collecting all rate constants In one term gives

Where KM is called the Michaelis constant It is difficult to determine the Vmax experimentally the
It is now possible to solve for the concentration of the enzyme- equation for a hyperbola
substrate complex, [ES]

Can be transformed into the equation for a straight line by

taking the reciprocal of each side

Or alternatively,

In the initial stages, formation of product depends only Lineweaver-Burk Plot

on the rate of breakdown of ES Has the form y=mx + b, ad is the formula for a straight line

If substrate concentration is so large that the enzyme is

saturated with substrate [ES] = [E]T
 A plot of 1/V versus 1/[S] will give a straight line with a slop of
KM/Vmax and y-intercept of 1/Vmax
Substituting k2[E]T = Vmax into the above equation  Such a plot is known as a Lineweaver-Burk double reciprocal
gives plot
 KM is the dissociation constant for ES; the greater the value of
KM, the less tightly S is bound to E
Vmax is the turnover number
 Generate the Michaelis-Menten plot

Significance of KM and Vmax Practice Session: Lineweaver-Burk Plot

 KM Plotting the results gives a straight line (best fit)
o Concentration of substrate when 50% of enzyme active
sites are occupied by substrate
o Dissociation constant for Es complex (on the assumption
that k-1 >> k2
o Low value for KM  high affinity for substrate
o High value for KM  low affinity for substrate
 Vmax
o Related to the turnover number, k2 also kcat
o Turnover number = Vmax/[ET] = kcat
o Number of moles of substrate that react to form the
product per mole of enzyme per unit time Practice Session: Results
o kcat
o A measure of enzyme efficiency

Turnover Numbers and KM

Plotting the results gives a straight line (best fit)
 Vmax = 1/y-intercept = 1/0.01 = 100 mM/min
 Slope = 0.01 min
 Slope = KM / Vmax
 KM = 1 mM

So, at 1mM substrate concentration, we expect the enzyme to

be 50% in the form of the ES complex

 KM Inhibition of Enzymes
o Concentration of substrate when 50% of enzyme active Irreversible inhibitor
sites are occupied by substrate A substance that causes inhibition that cannot be
reversed
Usually involves formation or breaking of covalent
bonds to or on the enzyme
Original enzyme cannot be regenerated
Reversible inhibitor
A substance that binds to an enzyme to inhibit it, but
can be released
Competitive inhibitor: binds to the active (catalytic) site
and blocks access to it by substrate
o If the substrate concentration is less than KM, then the Noncompetitive inhibitor: binds to a site other than the
enzyme is not activated. active site; inhibits the enzyme by changing its conformation
o If the substrate concentration is greater than KM, then the
enzyme is activated. Enzyme Inhibition
Competitive Inhibition
Practice Session Binds to the active site, preventing the substrate from
binding to the active site
Noncompetitive Inhibition
Uncompetitive Inhibition
Bind to the site other than the active site however upon
doing so, the shape of the active site cannot be anymore
complementary to the shape of the substrate or is not as
efficient as it should be
Mixed Inhibition

Competitive Inhibition
 Expected situation
 o There is an equilibrium between the free enzyme and the
enzyme-substrate complex
 Enzyme in equilibrium with a substrate from forming the ES
o Product is formed from the enzyme-substrate complex in
complex, however the enzyme can also bind to the inhibitor
the presence of a competitive inhibitor
but the binding of enzyme to the inhibitor does not prevent
the substrate from binding to the inhibitor itself, thus no
product can be formed
o However, the product can only be formed from the
enzyme substrate complex
 The maximum velocity VImax has the form
 There is an equilibrium between the free enzyme and the
enzyme-inhibitor complex
o Substrate competes with the inhibitor for the active site
o Product can only be formed from the ES complex
 Because the inhibitor does not interfere with the binding of
 Mechanism of Competitive Inhibition substrate to the active site, KM is unchanged
 Increasing substrate concentration cannot overcome
noncompetitive inhibition

 In the presence of a competitive inhibitor; the equation

changes

 In a Lineweaver-Burk double reciprocal plot of 1/V versus

1/[S], the slope (and the x-intercept) changes but thy-
intercept does NOT change
 Vmax is not changed
 Lineweaver-Burk Plot of an enzyme kinetics for competitive
inhibition

Uncompetitive Inhibition
 Inhibitor can bind to ES complex but NOT to free E

 Effect of competitive inhibitor can be overcome by increasing

[S]
 Competitive inhibitors usually resemble the substrate
o E.g. antidote for methanol poisoning is ethanol
o Increasing the amount of substrate can mitigate the effect
of inhibitor

Noncompetitive Inhibition
 Several equilibria are involved
 What is the effect?
o Vmax is decreased
o KM is decreased
 Result: Lineweaver-Burk plot of uncompetitive inhibitor is
PARALLEL to the uninhibited enzyme

Mixed Inhibition
 Inhibitor can bind through mix of the previous mechanisms
o Vmax is decreased
o KM is increased
 Lines for uninhibited and inhibited reactions intersect in the
second quadrant (not the x-axis and not the y-axis)

Practice Session

What did we expect to find out?

Michaelis-Menten Plot
 See if the enzymes follows Michaelis-Menten kinetics
(curve should be hyperbolic)
Lineweaver-Burk Plot
 If the enzyme follows Michaelis-Menten kinetics, we can
deduce the type of inhibition
o Lines intersect at y-axis -> competitive
o Lines intersect at x-axis -> noncompetitive
o Parallel lines -> uncompetitive
o Other -> mixed inhibition

Practice Session – Solution

Michaelis-Menten Plot

Lineweaver-Burk Plot