ENZYME PROPERTIES,
FUNCTIONS & KINETICS
Week 11: 23&24/11/2011
KHADIJAH HANIM ABDUL RAHMAN
SCHOOL OF BIOPROCESS ENGINEERING,
UNIMAP
SEM 1: 2011/2012
LEARNING OUTCOMES
 DISCUSS properties and classification of enzyme
 DISCUSS catalytic properties and enzymatic
regulation in cellular metabolism.
Introduction
 Most important functions of proteins- as catalysts
 Living processes consist almost entirely of biochemical
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reactions- without catalysts these reactions would not
occur fast enough.
Enzymes have several remarkable properties:
The rates of enzymatically catalyzed reactions are often
phenomenally high.
The enzymes are highly specific to the reactions they
catalyzed- side products are rarely formed.
Enzymes can be regulated- important consideration in
living organisms- must conserve energy and raw
materials.
 How do enzymes work?
- Recognize very specific substrates
- Perform specific chemical reactions at high speed.
- Enzymes make and break specific chemical
bonds of the substrates at a faster rate without
being consumed in the process.
- At the end of each catalytic cycle, the enzyme is
free to begin again with a new substrate molecule
Active site = a part of an enzyme where substrate bind and undergo a chemical reaction
Properties of enzymes
 Enzymes are generally globular proteins, some are
simple proteins and others are conjugated proteins.
 Catalysts- substance that enhances the rate of a
chemical reaction but not permanently altered by the
reaction.
 Catalysts decrease the activation energy required for
a chemical reaction – less energy.
 Transition state occurs at the apex of both reaction
pathway
 Reaction catalyze by enzyme require low activation
energy to convert reactant (substrate) from the
ground state to the transition state
Ground state
(stable, low energy
form of mol.)
 Each type of enzyme molecule contain a unique,
intricately binding surface called active site
 Substrate bind to active site which is typically small
cleft on a large protein mol.
 Beside being binding site, amino acid side chains
that line the active site actively participate in
catalytic process
LOCK & KEY Model
 Introduce by Emil Fischer 1890
 illustrate enzyme specificity
 Each enzyme binds to a single type of substrate (bcoz the
active site and the substrate have complementary
structure)
 active site = substrate binding surface
 The correct substrate fits into the active site of the enzyme
like a key into a lock. Only the right key fits.
INDUCED-FIT model
 Modification of lock and key model by Daniel Koshland
(1958)
 Flexible structure of protein is taken into account
 In this model, substrate does not fit precisely into a rigid
active site
 Noncovalent interaction between enzyme & substrate
change the active site structure, make it fit for the substrate
CLASSIFICATION OF ENZYMES
 Enzymes named according to the type of chemical
reaction it catalyzes
 Six major enzyme categories :
1) Oxidoreductase
- Catalyze oxidation-reduction (redox) reaction (enzyme
that catalyzes the transfer of electron from 1 molecule to
the other)
- Subclasses – dehydrogenases, oxidases, oxygenases,
reductases, peroxidases & hydroxylases
2) Transferases
- catalyze reactions that involve the transfer of groups from
one molecule to another
- eg. Transaminases, transcarboxylases
3) Hydrolases
- cleave bonds by adding water
- eg. Phosphatases, peptidases, esterase
4) Lyase
- catalyze reactions in which groups (eg. H20, CO2, NH3)
removed to form a double bond or added to a double
bond.
- eg. Decarboxylases, hydratases, deaminases
5) Isomerases
- catalyze intramolecular rearrangements. The epimerases
catalyze the inversion of asymmetric carbon atoms.
- Mutases catalyze the intramolecular transfer of functional
group.
- --- eg. epimerases or mutases
6) Ligases
- catalyze bond formation between two substrate
molecules
- eg. Synthetase, carboxylase
ENZYME KINETICS
 Enzyme kinetics is the quantitative study of enzyme
catalysis (chemical reaction catalyzed by enzyme).
 Kinetic studies measure reaction rates and the affinity of
enzymes for substrates and inhibitors
 Studying an enzyme's kinetics in this way can reveal the
catalytic mechanism of this enzyme, its role in metabolism,
how its activity is controlled, and how a drug or a poison
might inhibit the enzyme.
 The rate or velocity of a biochemical reaction is
defined as the change in the concentration of a
reactant or product per unit time.
 Another useful term to describe reaction- reaction’s
order.
 Order- determined empirically (experimentation).
 A reaction is said to follow first-order kinetics- rate
depends on the concentration of a single reactant.
 For a given enzyme concentration and for relatively low
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substrate concentrations, the reaction rate increases
linearly with substrate concentration
the enzyme molecules are largely free to catalyze the
reaction
increasing substrate concentration means an increasing
rate at which the enzyme and substrate molecules
encounter one another.
However, at relatively high substrate concentrations, the
reaction rate is zero-order with respect to substrate.
the enzyme active sites are almost all occupied
MICHAELIS-MENTEN KINETICS
 Models to investigate the enzyme rates
 Proposed by Leonor Michaelis and Maud Menten in
1913.
 When the substrate S binds in the active site of an
enzyme E, an intermediate complex (ES) is formed.
 During the transition state, the substrate is
converted into product.
 After sometimes, the product dissociates from the
enzyme.
 Rate equations for an enzyme catalyzed reaction support a
theory involving the formation of ES complexes.
 At high [S], S saturates E, and the reaction rate is
independent of the [S].
 The value of v under this condition is called the maximum
velocity, Vmax. At low [S], the reaction is first-order with
respect to S. The plot of v versus [S] from low to high [S] is
a rectangular hyperbola. The rate equation (MichaelisMenten equation) that describes this relationship is
 Vmax- the maximum velocity that the reaction can attain
 The concentration of substrate that corresponds to half-
maximum velocity is called the Michaelis constant, Km.
The enzyme is half-saturated when [S] = Km.
 Km is a measure of the substrate affinity for enzyme. A
small Km indicates high affinity- the rate will approach Vmax
more quickly.
 Km=small value, the enzyme can achieves max. catalytic
efficiency at low substrate conc- efficiency of enzyme to
convert substrate into product.
Michaelis-Menten
plot
Lineweaver-Burk Plots
 Km and Vmax values for an enzyme- determined by
measuring initial reaction velocities at various
substrate conc.
 Approximate values of Km and Vmax can be obtained
by constructing a graph.
 The Michaelis-Menten equation, hyperbola graph:
 Can be rearranged by taking it reciprocal:
 In Lineweaver-Burk plot- generated straight line,
y=mx + b ( y and x- 1/v and 1/[S], respectivelyvariables)
 m and b are constants ( Km/Vmax and 1/Vmax,
respectively)
 The intercept on the vertical axis is 1/Vmax
 The intercept on horizontal axis is -1/Km.
ENZYME INHIBITION
 Enzyme activity- can be inhibited
 Molecules that can reduced enzyme activity- inhibitors-
drugs, antibiotics, food preservatives and poisons.
 Investigation of enzyme inhibitors, important:
1) Enzyme inhibition- important means by which metabolic
pathways are regulated.
2) Numerous clinical therapies based on enzyme
inhibition.
- Eg: antibiotics and drugs reduce /eliminate the activity
of specific enzymes.
- The most effective AIDS treatment- multidrug therapy
that includes protease inhibitors- molecules that disable
a viral enzyme required to make new virus.
3) Enzyme inhibition- enable biochemist to develop
techniques to probe physical and chemical structure
and functional properties of enzymes.
 Enzyme inhibition occur when- a compound
competes with substrate for the active site of the free
enzyme.
 3 classes of enzyme inhibitors- competitive,
uncompetitive and noncompetitive inhibitors.
COMPETITIVE INHIBITORS
 Competitive inhibitors bind reversibly to free
enzyme, not the ES complex, to form an enzymeinhibitor (El) complex.
Substrate and inhibitor compete
for the same site on the enzyme
 Substance that behave as
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competitive inhibitors, reduce
enzyme affinity for substrate
Enzyme activity decline, no
reaction occur when EI complex
exists
Effect of competitive inhibitors on
enzyme activity is reversed by
increasing [S]
At high [S], all active site filled
with substrate and reaction
velocity reaches the value
observed without an inhibitor
Substances that behave as
competitive inhibitors- reduce the
enzyme’s affinity for substratesimilar in structure with
substrate.
With competitive inhibition- Vmax
stays constant, Km increases.
Shown in the double-reciprocal plot
as shift in horizontal intercept.
UNCOMPETITIVE INHIBITORS
 The inhibitors binds only to the enzyme-substrate
complex (ES), and not the free enzyme
 In uncompetitive inhibition- both Km and Vmax are
changed, although their ratio are still the same.
NONCOMPETITIVE INHIBITORS
 In some enzyme-catalyzed reactions, an inhibitors
can bind to both enzyme and enzyme-substrate
complex (EI)
 A noncompetitive inhibitor binds at a site other than
the active site of the enzyme
 Inhibitor binding result in a modification of
enzyme’s conformation that prevent product
formation-reduces the enzyme activity.
 Noncompetitive inhibitors do not affect substrate
binding & have little structural resemblance to
substrate
 Is only partially reversed by increasing the substrate
concentration
 In noncompetitive Vmax decreases and Km stays
constant
 The vertical intercept is shifted.
SUMMARY FOR ENZYME INHIBITION
COMPETITIVE
INHIBITION
UNCOMPETITIVE
INHIBITION
NONCOMPETITIVE
INHIBITION
-S & I cannot bind at the
same time- competing
for the enzyme’s active
site
- can be overcome by
increasing the [S]
- Vmax remain constant
-Km increase- it takes a
higher conc of S to reach
Km/half Vmax.
-I binds only to the (ES)
complex.
-Causes Vmax to decreaseas a result of removing
activated complex.
- Km decreases due to
better binding efficiency
and effective elimination
of the ES complex.
-Can bind to both enzyme
and ES complex.
-I binds to a site other
than the active site.
- binding of I to E reduces
its activity but does not
affect the binding of
substrate
-Vmax will decrease due to
the inability for the
reaction to proceed
efficiently
-Km will remain constant
as the actual binding of S
will still function
properly.
CATALYTIC MECHANISMS
 Enzyme catalysis is the catalysis of chemical reactions
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by specialized proteins known as enzymes
Despite extensive research, only a few of enzymes
mechanism in known.
Enzymes use the same catalytic mechanisms as
nonenzymatic catalysts.
Enzyme achieve higher catalytic rates because their
active site possess structure that uniquely suited to
promote catalysis
Factors contribute to enzyme catalysis :
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proximity and strain effects
electrostatics effects
acid-base catalysis
covalent catalysis
FACTOR INFLUENCING CATALYSIS
1) PROXIMITY AND STRAIN EFFECTS
 For a biochemical reaction to occur, the substrate must
closely approach the catalytic site with proper orientation
 Once substrate correctly positioned, result in a strained
enzyme-substrate complex.
 This strain help to bring the enzyme-substrate complex into
the transition state
 In general, the more tightly the active site can bind the
substrate while it is in the transition state, the greater the
reaction rate.
 When an enzyme and substrate are in very close proximity,
they behave as if they are part of the same molecule- it gives
the reaction intramolecular character- massive rate
increases.
 reactions occur faster if the reaction is intramolecular.
2) ELECTROSTATIC EFFECTS
 the strength of electrostatic interactions is related to the
capacity of surrounding solvent molecules to reduce the
attractive forces between chemical groups.
 Because water is largely excluded from the active sites as
the substrate binds, the dielectric constant near the active
site is often low, this may influence the chemical reactivity
of the substrate.
 weak electrostatic interactions, such as those between
permanent and induced dipoles in both the active site and
the substrate are believed to contribute to the catalysis.
 A more efficient binding of substrate lowers the free energy
of transition state- accelerates the reaction.
3) ACID-BASE CATALYSIS
 Chemical groups can often be made more reactive by
adding or removing a proton.
 Enzyme active sites contain side chain groups that
act as proton donors or acceptors.
 These groups referred as- general acids and general
bases (substance that can release proton/accept
proton, respectively)
 General acids/base- release/accept proton and
contribute to reaction rate acceleration.
4) COVALENT CATALYSIS
 In some enzymes a nucleophilic side chain group forms an
unstable covalent bond with the substrate
 The enzyme-substrate complex then forms product
 The covalent bond must, at a later stage in the reaction, be
broken to regenerate the enzyme.
 This mechanism is found in enzymes such as proteases like
chymotrypsin and trypsin, where an acyl-enzyme
intermediate is formed.
Serine proteases
R-C-NH-R’
+
Enzyme-CH2OH
R’-NH2
R-C-O-CH2-Enzyme
H2O
R-C-OH
+
Enzyme-CH2OH
 Serine proteases- Uses the –CH2OH group as a
nucleophile to hydrolyze peptide bonds
The role of cofactors in Enzyme catalysis
 Catalytic activity of some enzymes depends only on
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interaction between active site amino acids and substrate
other enzymes require non protein compounds for their
activities (cofactor)
Enzyme cofactors- ions such as Mg2+ /Zn2+ , or
Complex organic molecules referred to as coenzyme
Apoenzyme = protein component of an enzyme that lacks
an essential cofactor
Holoenzyme = intact enzyme with bond cofactor
Cofactors: METALS
 2 classes of metals- transition metals (Fe2+ & Cu2+ )
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- alkali & alkaline earth metals
(Na+, K+, Mg2+ and Ca2+ )
Transition metals- the most often involved in catalysiselectronic structures.
Metal ions provide a high concentration of +ve charge, is
useful in binding small molecules
Transition metals act as lewis acid (electron pair
acceptors)- effective electrophiles (amino acid side chainspoor electrophiles- cannot accept unshared pairs of
electrons).
Because their directed velences allow them to interact with
2/ more ligands, metal ions help to orient the substrate
within the active site.
The substrate-metal ion complex polarizes the substrate
and promotes catalysis
Coenzymes
 Coenzymes are derived by vitamins
 Vitamins- organic nutrients required in small
amounts in human diet.
 2 classes – water-soluble
- lipid-soluble
 Vitamin-like nutrients (eg. Lipoic acid, carnitine)
that can be synthesized in small amounts, facilitate
enzyme-catalyzed reaction
Vitamins & their coenzyme forms
Vitamin
Water Soluble
vitamin
Thiamine (B1)
Riboflavin (B2)
Pyridoxine
Nicotinic acid
Lipid soluble
vitamins
Vitamin A
Vitamin D
Coenzyme form
Reaction
Thiamine pyrophospophate
FAD and FMN
Pyridoxal phosphate
NAD and NADP
Decarboxylation
Redox
Amino group transfer
Redox
Retinal
Vision, growth
1,25-Dihydroxycholecalciferol Calcium and
phosphate metabolism
Exercises
 Identify each of the following as a cofactor,
coenzyme, apoenzyme or holoenzyme:
a) Zn2+
b) Active alcohol dehydrogenase
c) Alcohol dehydrogenase lacking Zn2+
d) FMN
e) NAD+
Effects of Temperature and pH on enzyme
catalyzed reactions
 Any environmental factor that disturbs protein
structure may change enzymatic activity
 Enzymes are especially sensitive to changes in temp
and pH.
Temperature
 Chemical reactions- affected by temperature
 The higher the temperature, the higher the reaction
rate
 Reaction velocity increases- becoz, more molecules
have sufficient energy to enter transition state.
 The rate of enzyme-catalyzed reaction also increase
with increasing temp.
 However, enzymes are protein that denatured at
high temperature
 Each enzymes has an optimum temperature at which
it operate at maximal efficiency
 If temperature raised beyond it optimum
temperature, enzyme activity will declines
 An enzyme’s optimum temp usually close to the
normal temp. of an organism it comes from
pH
 Hydrogen ion concentration effects enzymes in several
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ways
First, catalytic activity is related to ionic state of active
site
Changes in [H] ion can effect the ionization of active
site groups.
Eg: Catalytic activity of certain enzymes require
protonated form of side chain amino group
If the pH become sufficiently alkaline, the group will
lose it proton, enzyme activity will depressed
Eg. If a substrate contains an ionizable group, a change
in pH may alter its capacity to bind to the active site.
Changes in ionizable groups may change the tertiary
structure of the enzyme.
Drastic changes in pH often lead to denaturation
 Few enzymes can tolerate large changes in pH, but
most enzymes are active only within a narrow pH
change
 pH value at which enzyme’s activity is max is called
pH optimum