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Outline of Enzyme
Introduction
Enzyme kinetics
- Mechanistic models for simple enzyme kinetics
- Effect of pH and Temperature
Immobilized Enzyme System
- Method of immobilization
- Diffusional limitations
- Electrostatic and steric effects
Large-scale production and application of
enzyme.
What is Enzyme?
An enzyme is a protein molecule that is a biological
catalyst with the following characteristics.
• Effective to increase the rate of a reaction.
Most cellular reactions occur about a million times faster
than they would in the absence of an enzyme.
• Specifically act with one reactant (called a substrate) to
produce products.
maltase that converts maltose to glucose
• Be regulated from a state of low activity to high activity and
vice versa.
e.g. some enzyme activity is inhibited by the product.
• Be versatile: More than 3000 enzymes are identified
Enzyme
Enzyme has high molecule weight (15,000< mw<
several million Daltons).
Holoenzyme is an enzyme contains non-protein
group.
Such non-protein group is called cofactor such as
metal ions, Mg, Zn, Mn, Fe
Or coenzyme, such as a complex organic
molecule, NAD, or vitamins.
Apoenzyme is the protein part of holoenzyme.
Holoenzyme = apoenzyme + cofactor (coenzyme)
Enzyme nomenclature
Enzyme is named by adding the suffix –ase
- to the end of the substrate that is to be converted
to the desired product.
e.g. urease that changes urea into ammonium carbonate.
protease that converts protein or polypeptides to
smaller molecules such as amino acids.
- to the reaction catalyzed.
e.g. phosphoglucose isomerase that converts glucose-6phosphate to fructose-6-phosphate.
Alcohol dehydrogenase that catalyzes the removal of
hydrogen from alcohol.
Enzyme classification
International Classification of Enzymes
by the International Classification Commission in
1864.
Enzymes are substrate specific and are classified
according to the reaction they catalyze.
Enzyme Nomenclature, 1992, Academic Press, San Diego,
California, ISBN 0-12-227164-5.
http://www.chem.qmul.ac.uk/iubmb/enzyme/
Enzyme classification
Enzymes can be classified into six main classes:
- Oxidoreductases: catalyze the oxidation and reduction
e.g. CH3CH2OH → CH3CHO+H+
- Transferases: catalyze the transfer of a functional group (e.g.
a methyl or phosphate group) from one molecule (called the
donor) to another (called the acceptor).
A–X + B → A + B–X
- Hydrolases:catalyze the hydrolysis of a chemical bond.
A–B + H2O → A–OH + B–H, e.g. peptide bond
Enzyme classification
- Lyases:catalyze the breaking of various chemical bonds
by means other than hydrolysis and oxidation, often
forming a new double bond or a new ring structure
e.g. CH3COCO-OH → CH3COCHO (dehydratase)
- Isomerases:catalyse the interconversion of isomers.
e.g.phosphoglucose isomerase that converts glucose-6phosphate to fructose-6-phosphate.
- Ligases:catalyse the joining of two molecules by forming a
new chemical bond, with accompanying hydrolysis of
ATP or other similar molecules
ATP + L-tyrosine + tRNATyr = AMP + diphosphate +
L-tyrosyl-tRNATyr
Enzyme classification
Each main class contains subclasses, subsubclasses and
subsubsubclasses. There are four levels of classes in total.
e.g.
Reaction: CH2CH2OH+NAD+→CH3CHO (acetaldehye)+NADH+H+
Systematic name: alcohol NAD oxidoreductase (1.1.1.1)
Trivial name: alcohol dehydrogenase
1. Oxidation-reduction reaction
1.1 Acting on R-OH group of substrates
1.1.1 Requires NAD+ or NADP+ as hydrogen acceptor
1.1.1.1 Specific substrate is ethyl alcohol
Mechanism of enzyme catalysis
What is a catalyst?
• A catalyst is a substance that accelerates the
rate (speed) of a chemical reaction without itself
being consumed or transformed.
• It participates in reactions but is neither a
chemical reactant nor chemical product.
S
P
S + C (catalyst)
P + C (catalyst)
Mechanism of enzyme catalysis
• Catalysts provide an alternative pathway of lower
activation energy for a reaction to proceed whilst
remaining chemically unchanged themselves.
Free energy change
Mechanism of enzyme catalysis
• Catalysts lower the activation energy of the
reaction catalyzed by binding the substrate and
forming an catalyst-substrate complex which
produces the desired product.
• Catalysts lower the activation energy of the
catalyzed reaction, but does not affect free
energy change or equilibrium constant.
Mechanism of enzyme catalysis
The reaction rate v is strongly affected by the
activation energy of the reaction.
v = k*f(S)
-f(S) denotes the function of substrate concentration
-k is the rate constant which can be expressed by
Arrhenius equation (H.S. Fogler, Chemical Reaction Engineerng,
Prentice-Hall Inc., 2005)
k=A*exp(-Ea/R*T)
A is a constant for a specific system,
Ea is the activation energy,
R is the universal gas constant, and T is the temperature (in
degrees Kelvin).
When Ea is lowered, k is increased, and so is the rate.
Mechanism of enzyme catalysis
•
Catalysts do not affect free energy
change or equilibrium constant of the
catalyzed reaction.
Free energy (G) is the energy stored in the bonds
of a chemical that can be harnesses to do
work.
Free energy change (ΔG) of a reaction refers to
the change between the free energy in the
product (s) and that in the substrate(s).
Mechanism of enzyme catalysis
For an example,
S
P
S + C (catalyst)
P + C (catalyst)
• For uncatalyzed reaction,
free energy change ΔG, uncatalyzed=G(P)-G(S)
• For catalyzed reaction,
free energy change ΔG, catalyzed=G(P)-G(S)
Therefore,
ΔG, uncatalyzed= ΔG, catalyzed
Mechanism of enzyme catalysis
S
P
S + C (catalyst)
P + C (catalyst)
• Free energy change determines the reaction equilibrium –
the maximum amounts of the product could be
theoretically produced.
• Reaction equilibrium is represented by reaction equilibrium
constant Keq,=γp[P]/ γs[S]
- ΔG, uncatalyzed=RTln Keq
[ ] represents the concentration of the compounds.
γp and γs are activity constants of the product and the
substrate, respectively.
Mechanism of enzyme catalysis
• Catalysts can not increase the amounts of the
product at reaction equilibrium.
• Catalysts can only accelerate the reaction rate to
reach the reaction equilibrium.
Mechanism of enzyme catalysis
• Enzymes are catalysts and have the
characteristics of common catalysts.
Particularly,
• Enzymes are
- Efficient
- Specific
- regulated
- versatile
Efficiency of Enzyme Catalysis
For an example, in the reaction of
decomposition of hydrogen peroxide, the
activation energy Ea,o of the uncatalyzed
reaction at 20oC is 18 kcal/mol, whereas
that for chemically catalyzed (Pt) and
enzymatically catalyzed (catalase)
decomposition are 13 kcal/mol (Ea,c) and
7 kcal/mol (Ea, en), respectively.
Compare the reaction rates at these three
different conditions.
Efficiency of Enzyme Catalysis
2H2O2
2H2O + O2
The forward reaction rate r (moles/L-s),
r = k*f(H2O2)
k is the forward reaction rate constant,
f(H2O2) is a function of substrate perioxide
concentration.
If k is represented by k0, kc, and ken , respectively,
for the uncatalyzed, chemically catalyzed (Pt)
and enzymatically catalyzed reactions, the
corresponding reaction rates (r0, rc, ren) can be
expressed as follows
r0 = k0*f(H2O2)
rc = kc*f(H2O2)
ren = ken*f(H2O2)
The ratio of the chemically catalyzed rate to the
uncatalyzed rate can be calculated by as follows:
rc /r0 = kc*f(H2O2)/k0*f(H2O2)
Similarly, The ratio of the enzymatically catalyzed rate to
the uncatalyzed rate can be calculated by as follows:
ren /r0 = ken*f(H2O2)/k0*f(H2O2)
At 20oC, with the same initial concentration of reactant
hydrogen peroxide, f(H2O2) remains the same for the
above three process at initial conditions, the initial
reaction rates vary with the rate constants.
rc /r0 = kc/k0
ren /r0 = ken/k0
Using Arrhenius Equations yields,
rc /r0 = kc/k0 = (A*exp(-Ea,c/R*T))/(A*exp(-Ea,o/R*T))
ren /r0 = ken/k0 = (A*exp(-Ea,en/R*T))/(A*exp(-Ea,o/R*T))
Where Ea,o, Ea,c and Ea,en are activation energy for
uncatalyzed reaction, chemically catalyzed and
enzymatically catalyzed reactions, respectively.
A is a constant and remains same for the specific system,
the rate ratios could be simplified to:
rc /r0 = (exp(-Ea,c/R*T))/(exp(-Ea,o/R*T))
ren /r0 = (exp(-Ea,en/R*T))/(exp(-Ea,o/R*T))
When T= 20oC = 273+20 (K) = 293 K,
Ea,o = 18 kcal/mol, Ea,c = 13 kcal/mol (Ea,c) and
Ea, en =7 kcal/mol, respectively.
R = 8.314 J mol-1K-1 , 1 cal = 4.18 J.
Then substituting the parameters in the rate ratio eqns. with
these values, yields,
rc /r0 = (exp(-Ea,c/R*T))/(exp(-Ea,o/R*T))
= (exp(-13000*4.18/8.314*293))/(exp(-18000*4.18/8.314*293))
=?
ren /r0 = (exp(-Ea,en/R*T))/(exp(-Ea,o/R*T))
=(exp(-7000*4.18/8.314*293))/(exp(-18000*4.18/8.314*293)
=?
When T= 20oC = 273+20 (K) = 293 K,
Ea,o = 18 kcal/mol, Ea,c = 13 kcal/mol (Ea,c) and
Ea, en =7 kcal/mol, respectively.
R = 8.314 J mol-1K-1 , 1 cal = 4.18 J.
Then substituting the parameters in the rate ratio eqns. with
these values, yields,
rc /r0 = (exp(-Ea,c/R*T))/(exp(-Ea,o/R*T))
= (exp(-13000*4.18/8.314*293))/(exp(-18000*4.18/8.314*293))
=5.1X103
ren /r0 = (exp(-Ea,en/R*T))/(exp(-Ea,o/R*T))
=(exp(-7000*4.18/8.314*293))/(exp(-18000*4.18/8.314*293)
=1.4X108
Enzyme catalysis is efficient!
- If it takes 1 hr to complete the reaction with
enzyme
- it needs to take
1.4X108 hrs = 5833333 days = 15981years
to complete the same reaction without enzyme
catalysis.
Specificity of Enzyme Catalysis
• Much of the catalytic power of enzymes comes from their
bringing substrates together in favorable orientations to
promote the formation of the transition states in enzymesubstrate (ES) complexes.
E +S
ES
E +P
• The substrates are bound to a specific region of the
enzyme called the active site.
• Most enzymes are highly selective in the substrates that
they bind. The catalytic specificity of enzymes depends
in part on the specificity of binding.
Specificity of Enzyme Catalysis
Lock-and-Key Model of Enzyme-Substrate Binding
(Emil Fischer ,1890)
.
In this model, the active site of the unbound enzyme
is complementary in shape to the substrate
The Active Sites of Enzymes Have
Some Common Features
The active site of an enzyme is the region that
binds the substrates (and the cofactor, if any).
• It also contains the residues that directly
participate in the making and breaking of bonds.
These residues are called the catalytic groups.
• The interaction of the enzyme and substrate at
the active site promotes the formation of the
transition state.
The Active Sites of Enzymes Have
Some Common Features
• The active site is a three-dimensional cleft
formed by groups that come from different parts
of the amino acid sequence.
• The active site takes up a relatively small part of
the total volume of an enzyme.
The "extra" amino acids serve as a scaffold to create the
three-dimensional active site from amino acids that are
far apart in the primary structure.
• Substrates are bound to enzymes by multiple
weak attractions
Interactions in ES complexes are much weaker than
covalent bonds.
The Active Sites of Enzymes Have
Some Common Features
• The specificity of binding depends on the precisely
defined arrangement of atoms in an active site.
- The lock-and –key model (Emil Fischer):
The enzyme has a fit shape before the substrate
is bound.
- The Induced-Fit Model (Daniel Koshland, Jr. 1958)
Enzymes are flexible and the shapes of the active
sites can be markedly modified by the binding of
substrate.
Induced-Fit Model of Enzyme-Substrate Binding.
In this model, the enzyme changes shape on substrate binding.
The active site forms a shape complementary to the
substrate only after the substrate has been bound.
Summary of Introduction
• Enzyme classification
• Enzyme have common catalytical features
- decrease the reaction activation energy
- does not affect equilibrium
• Enzyme special catalytic features
- Efficient
- Specific
- regulated
- versatile
Enzyme kinetics
Michaelis-Menten kinetics or saturation kinetics
which was first developed by V.C.R. Henri in 1902 and
by L. Michaelis and M.L. Menten in 1913.
This model is based on data from batch reactors
with constant liquid volume.
- Initial substrate, [S0] and enzyme [E0]
concentrations are known.
- An enzyme solution has a fixed number of
active sites to which substrate can bind.
- At high substrate concentrations, all these sites
may be occupied by substrates or the enzyme is
saturated.
Saturation Enzyme Kinetics
Enzyme kinetics
E.g. a simple reaction scheme that involves a reversible step
for enzyme-substrate complex formation and a
dissociation step of the ES complex.
K1
E+S
K-1
k2
ES  P  E
where the rate of product formation v (moles/l-s, g/l-min) is
d[ P]
v
 k 2 [ ES]
dt
Ki is the respective reaction rate constant.
Enzyme kinetics
The rate of variation of ES complex is
d[ES]
 k1[E][S ]  k1[ES]  k2[ES]
dt
Since the enzyme is not consumed, the
conservation equation on the enzyme
yields
[ E]  [ E0 ]  [ ES]