Enzymes and their Regulations

Lecture 2

Prof. Chandrani Wijeyaratne

B.Sc. (Hons.), M. Phil , Ph.D (Kyushu, Japan),
Post-graduate Dip. in Microbiology (Osaka, Japan
 Enzymes
• Definition catalyzes reaction but is not itself consumed
during the process
• Reactions in living organisms are catalized by protein
molecules - Enzymes are proteins of high molecular wt.
• Catalytic machinery of living systems
• “Enzyme “ comes from Latin word, : en, in; zyme, yeast
which means “in yeast” & closely associated with yeast
activity.
• Fermentative activity of yeast was discovered by Louis
Pasteur.
• Alcohol precipitate of malt extract contained
thermostable substance which converted starch into
sugar –first recognition of enzymes .It was called diastase
 History of Microbial Enzymes
• Use of rennin isolated from calf stomach in cheese making
was 1st. application of cell free enzymes
• Rennin, aspartic protease & coagulates milk protein
• Trypsin enzyme isolated from animals degraded proteins &
used as a detergent
• In 1959 introduced bacterial Bacillus protease
• Pectinase enzyme was used clarify fruit juices
• Microbial enzymes in food industry started in 1960s in
starch industry. Traditional acid hydrolysis of starch was
completely replaced by alpha amylase ands and
glucoamylases which could convert starch with over 95%
to yield glucose
 Microbial Enzymes- Present status
• At present enzymes are used for wide variety of
applications –main industries are:
• Detergents (37%)
• Textile (12%)
• Starch (11%)
• Baking (8%)
• Animal feed (6%) &
• Medicine
• Enzymes are used in biocatalytic processes involving
living or dead and permeabilized microorganisms
• Through genetic , protein and pathway engineering
techniques chemical are produced by living cells
 Advantages of Microbial enzymes
• Have the ability to function under mild
conditions of temperature, pH and pressure
• Therefore consume less energy
• Corrosive- resistant expensive equipment not
required
• Enzymes are specific and do not produce
unwanted by products
• Extensive refining and purification of target
product not required
• Processes are environmentally friendly
 Nomenclature
• Many enzymes are named by adding the suffix –ase to
the name of the substrate
• E.g. Urease catalyzes hydrolysis of urea
• Arginase catalyzes hydrolysis of arginine
• Some enzymes do not denote their substrates e.g.
pepsin and trypsin
• Sometime one and the same enzyme is known by two
or more names or different enzymes given the same
name.
• Because of these and other ambigities a systematic
basis for naming and classifying enzymes have been
adopted
 Enzyme classification
• Enzymes are classified on the basis of the reaction they
catalyze
• This system places all enzymes in six major classes, each
with subclasses, based on the type of reaction
catalyzed
• Each enzyme is assigned a four-digit classification
number and a systematic name, which identifies the
reaction catalyzed
 International classification of Enzymes
• It is based on the reaction they catalyze

No Class Type of reaction catalysed

1 Oxidoreductase Transfer of electrons

2 Transferases Group –transfer reactions

3 Hydrolases Hydrolysis reactions

Transfer of functional groups to water
4 Lyases Addition of groups to double bonds or the
reverse
5 Isomerases Transfer od groups within molecules to yield
isomeric forms
6 Ligases Formulation of C-C, C-S, C-O and C-N bonds
by condensation reactions coupled to ATP
cleavage
 Enzyme classification
E.g. ATP + D-glucose ADP+D-glucose 6
Phosphate
• The formal systematic name of this enzyme is
ATP:glucosephosphotransferase which indicates that it
transfer Phosphate gp from ATP to glucose
• It is in class 2 & classification number -2.7.1.1
Where the 1st. digit (2) stands for the class name
(transferase), 2nd.digit(7) for the subclass
(phosphotransferases), the 3rd. digit (1) for sub-subclass
(phosphotransferases with hydroxyl gp as acceptor and
the 4th digit (1) for D-glucose as the phosphate gp.
Acceptor. When the systematic name is cumbersome
trivial name may be used. Here it is hexokinase
 Properties of Enzymes
• Usually Proteins of high molecular weight (15,000< MW< several
million daltons)
• Some RNA moleculaes also catalytic – Ribozyme
• Enzymes are specific, versatile & effective biological catalysts
resulting in high reaction rates under ambient conditions
• Some have a simple structure –folded polypeptide chain
• Many enzyme have more than one subunit
• Some protein enzymes require a nonprotein gp for their activity.
This gp. is either a cofacter, such as, Mg, Zn, Mn,Fe or a coenzyme
such as complex organic molecule, NAD, FAD, CoA or some
vitamins
• An enzyme containing a nonprotein gp is called holoenzyme,
Protein part is apoenzyme
• Holoenzyme =apoenzyme +cofactor
• Isoenzyme –different molecular form but catalyze same reaction
 Mode and mechanism of enzyme action
• Enzymes lower the activation energy of the reaction
catalyzed by binding the substrate & forming enzyme-
substrate complex
• Enzymes do not affect the free-energy change or the
equilibrium constant
 Mode and mechanism of enzyme action
• For a reaction to occur, reactant molecules must contain
sufficient energy to cross a potential energy barrier, the
activation energy.
• All molecules possess varying amounts of energy
• Generally, only a few have sufficient energy for reaction.
• The lower the potential energy barrier to reaction, the more
reactants have sufficient energy and, hence, the faster the
reaction will occur.
• When the potential energy barrier is too high, and less reactants
have sufficient energy , slower the reaction
2) Activation Energy and the Reaction Coordinate

Transition State Theory: developed in 1930s

HA-HB + HC --- HA + HB-HC
Transition state: HA--HB—HC

Transition state = point of highest free energy

= most unstable
Reactants approach one another along a path of
minimal free energy = reaction coordinate
Transition state diagram/reaction coordinate diagram:
Plot of free energy versus the reaction coordinate
 Transition state diagram
Symmetrical

Transition
State

Substrate Product
Transition state diagram
Asymmetrical

Free energy
of activation

Free energy
of reaction
A simple enzymatic reaction might be written

where E, S, and P represent the enzyme, substrate, and product, respectively.

ES and EP are complexes of the enzyme with the substrate and with the product, respectively

A reaction coordination diagrams with and with out enzymes

 Activation Energy and the
Reaction Coordinate
• Activation energy – Amount of energy in calories
required to bring all the molecules in 1mol of a substance
at a given temperature to the transition stage at the top
of the energy barrier
• The greater the free energy of activation,
the slower the reaction rate
• If the free energy of the reaction, ΔG<0,
then the reaction is spontaneous and
releases energy (heat)
All catalysts, including enzymes, function by forming a
transition state, with the reactants, of lower free energy
than would be found in the uncatalysed reaction
(e.g. the activation energy for the uncatalysed
breakdown of hydrogen peroxide to oxygen and water is
76 kJ M-1 whereas, in the presence of the enzyme
catalase, this is reduced to 30 kJ M-1 and the rate of
reaction is increased by a factor of 108, sufficient to
convert a reaction time measured in years into one
measured in seconds)
 Mode of Action

Substrate fits in the enzyme active site,

just like a key fits into a lock.

An enzyme-substrate complex is formed.

Chemical reactions occur at the active site and

products are formed.
 catalase
Hydrogen peroxide  water and oxygen
What are the factors
affecting Enzyme
Activity?
 0°C
• Low temperatures  low Kinetic Energy
of enzymes and substrates.

• No/Very few enzyme-substrate

complexes are formed.

• Enzymes are inactivated.

20°C (increasing temperature)
• Increasing the temperature will lead to the increase
in kinetic energy of enzyme and substrate molecules.

• Enzyme and substrate molecules move with

increasing speed and collide more frequently with
each other.

• This increases the rate of enzyme-substrate complex

formation This increases the rate of enzyme-
substrate complex formation and product
formation.
Rate of reaction
increases
 37°C
• As the temperature continues to
increase, the rate of enzyme activity also
increases until the optimal temperature
is reached.

• Optimal temperature is the temperature

at which the enzyme works best. Rate of
product formation is highest!
 Beyond Optimal Temperatures
• At high temperatures (>60°C), weak bonds
within the enzyme molecule are broken
• Enzyme loses its shape and its active site.
• Loss of shape leads to a loss of function.
Enzyme is said to have denatured
• Denaturation is the change in 3D structure of
an enzyme or any other protein caused by
heat or chemicals such as acids or alkali,
causing it to lose its function.
 Denaturation

Different enzymes denature at different temperatures. Most enzymes denature at

temperatures higher than 60°C. However, there are some enzymes that stay active even
at high temperatures like 80°C (Enzymes in the bacteria Thermus aquaticus)
 Effect of pH on enzyme activity
• Enzyme works best within a narrow pH
range
• Each enzyme works best at particular pH,
known as its optimum pH level.
• At extreme pH levels, enzymes lose their
shape and function and become
denatured.
Effect of pH on enzyme activity
Effect of Substrate on Enzyme Activity
 Effect of Substrate on Enzyme Activity

Because the curve expressing this relationship has

the same general shape for most enzymes
(rectangular hyperbola) Michaelis and Menten
defined a constant designated as Km , that is useful
in establishing relationship between the substrate
concentration and the velocity of the enzyme-
catalyzed reaction.
Michaelis –Menten constant – Concentration of
the specific substrate at which a given enzyme
yields one-half its maximum velocity.
 Enzyme regulation
Regulate: to control or direct according to a rule ,
principle or law
Enzyme regulation : is the control of the rate of
reaction catalyzed by some effector (by inhibitors
or activators) or by alteration of some of condition
(e.g. by pH or ionic strength)
 Regulatory enzymes
In cell metabolism, groups of enzymes work together in
sequential chains or systems to carry out a given
metabolic process.
e.g. conversion of glucose into lactic acid in skeletal
muscle or synthesis of AA from simple precursors. In such
enzymes systems the reaction product of the first enzyme
becomes the substrate of the next and so on.
In each enzyme system there is at least one enzyme , the
‘pacemaker” that sets the rate of the overall sequence
because it catalyses the slowest or rate limiting step.
Often the first enzyme of a multi enzyem systems is the
pacemaker enzyme.
Five ways by which enzymes activity in the cell can be
changed

1. Enzyme production – Synthesis or degradation

2. Compartmentation – Different metabolic pathway occur in
different cell compartments
3. Activation or inhibition – by activators or inhibitors for e.g. feed
back inhibition by one of the products of the reaction
4. Post-translational modification – for e.g. by phosphorylation,
glycosylation or methylation
5. Localization to a different environment - ( from reducing
(cytoplasm) to an oxidizing (periplasm), to a low pH to a high pH,
low salinity to high salinity , high to low energy charge
 Enzyme Regulation

Constitutive enzymes- Enzyme needed at the

same level at all of the time

Regulated enzymes- Enzymes needed under some

conditions but not others for e.g. enzymes of the
Lac Operon
Enzymes are made to break down lactose only if
lactose is present
 Types of Regulation
Regulation of the amount of enzymes made
- At the level of transcription- RNA made
- At the level of translation- protein made
- Slower process -
Regulation of enzyme activity
- After the protein is synthesized
- Post translational modification
- Very rapid process
 Regulatory enzymes
Pacemaker enzymes ,whose activity is modulated through
various types of molecular signals are called regulatory
enzymes
Two Major classes of regulatory enzymes:
Allosteric or noncovalently regulated enzymes
Covalently regulated enzymes
 Allosteric enzymes
In some multienzyme systems the first or the regulatory
enzyme is inhibited by the end product of the multienzyme
system. When the end product increases above its usual
steady state concentration, the end product acts as a
specific inhibitor of the regulatory /first enzyme in the
sequence.
E-1- Threonine dehydralase
L-Threonine A B C D L- Isoleucine
E-2 E-3 E-4 E-5

The whole enzyme system slows down to bring the rate of

productioon of its end product back into balance with cell’s needs.
This type of regulation is called feedback inhibition .
 Allosteric enzymes
Allosteric feedback inhibition first discovered in bacterial
enzyme system that catalyses L-Threonine to L-Isoleucine .
There are five enzymes and the first Threonine dehydratase
is inhibited by Isoleucine the product of the last enzyme.
• Isoleucine is quite specific as an inhibitor
• Inhibition of Theronine dehydratase by isoleucine is
reversible; if isoleucine concentration decreases, the rate of
threonine dehydratase activity increases
• Thus threonine dehydratase activity responds very rapidly
& reversibly to fluctuations of isoleucine concentration in
the cell.
•Though Isoleucine specific inhibitor, does not bind to its
substrate site, it binds to regulatory site on the enzyme
 Allosteric enzymes
• Binding of isoleucine to the regulatory site of threonine
dehydratase is noncovalent and thus is readily reversible
• Threonine dehydratase is a typical allosteric
regulatory enzyme that function through
reversible, noncovalent binding of a modulator
molecule.
•Allosteric derives from Greek allo, “other”, and
stereos, ”space” or site.
•Allosteric enzymes are those having “other sites”.
 Properties of Allosteric enzymes
• Like all enzymes have an catalytic sites which bind the
substrate and transform it
• They have one or more regulatory or Allosteric sites for
binding the regulating metabolite, which is called effector
or modulator.
• Allosteric site is specific for its modulator
•These molecules are generally larger & more complex than
those of simple enzymes.
• Most of them have two or mor polypeptide chains or
subsubits
•They show significant deviation from classical Michaelis-
Menton behaviors
 Importance of Allosteric enzymes
1. Control of Metabolism:Cellular needs and conditions vary
from cell to cell and change within individual cells over time. For
example, stomach cells require different amounts of energy than
skin cells, fat storage cells, blood cells, and nerve cells. The same
stomach cell may also need more energy immediately after a meal
and less energy between meals.
 Covalently regulated enzymes
These are modulated through inter-conversion of their
active and inactive forms by covalent modification of the
enzyme molecule
E.g. Regulatory enzyme glycogen phosphorylase of muscle
and liver, which catalyses the reaction:
(Glucose)n +Phosphate (glucose)n-1 + glucose 1-phosphate
Glycogen
Glycogen phosphorylase occurs in 2 forms, the active form
phosphorylase a and the relatively inactive form phosphorylase b.
The phosphate group can be hydrolytically removed from
phosphorylase a by an enzyme called Phosphorylase phosphatase
Phjosphorylase a + 2H2O Phosphorylase b + 2Pi
 Covalently regulated enzymes
In this reaction phosphorylase a is converted to
phosphorylase b which is less active than Phosphorylase a
in catalyzing glycogen breakdown
This conversion of active form of the enzyme to relatively
inactive form is due to cleavage of two covalent bonds
between phosphoric acid and the two specific serine
residue in the enzyme
Phosphorylase b can in turn be reactivated i.e covalently
transdormed back into active phosphorylase a , by
another enzyme, phosphorylase kinase which calatyzes
transfer of phosphate gps from ATP to the hydroxyl groups
of the specific serine residues in phosphorylase b
 Cofactors and Coenzymes
Some enzymes require for activity an additional
chemical component called a cofactor.
Cofactor may be either inorganic, such as Fe+2,
Mn2+, or Zn2+ ions or it may be a complex organic
molecule called a coenzyme
Some enzyme require both coenzyme and one or
more metal ions for activity.
In some enzymes the coenzyme or metal ion is
only loosely and transiently bound , in which case
it is called a prosthetic group.
 Coenzyme
A complete, catalytically active enzymes together with its
coenzyme or metal ion is called a holoenzyme
Coenzyme and metal ions are heat stable whereas the the
protein part of an enzyme called the apoenzyme is
denatured by heat

Some enzyme containing or requiring essential inorganic elements

as cofactors
Fe+2, Fe+3, Cytochrome oxidase, Catalase, Peroxidase
Cu2+ , Cytochrome oxidase
Mg2+, Hexokinase, Glucose 6- phosphate
Zn2+ ions DNA polymerase, Alcohol dehydrogenase
 Competitive Inhibition
Most enzymes can be poisoned or inhibited by certain
chemicals reagents.
Information with regard to substrate specificity, the
nature of the functional gps at the active site, &
mechanism of the catalytic activity has been obtained
from studies on enzyme inhibition
Two major types of inhibitors : Reversible and Irreversible
Irreversible inhibitors: those that combine with or
destroy a functional gp on the enzyme molecule that is
needed for its catalytic activity
e.g.diisopropylfluorophosphate (DEP) which inhibits
acetylecholinesterase important in transmission of nerve
impulses. It catalyzers hydrolysis of acetylecholine.
 Reversible inhibitors
There are two kinds of reversible inhibitors:
Competitive and Non-competitive
Competitive inhibitor competes with the substrate for
binding to the active site but once bound cannot be
transformed by the enzyme
It can not be reversed or relived simply by increasing the
substrate concentration
Competitive inhibitor usually resemble the normal
substrate in three dimensional structure
e.g. malonate anion inhibits succinate dehydrogenase,
which removes 2 hydrogen atoms from succinate
When succinate contn. is increased extent of inhibition get
reduced
 Non-competitive Inhibition
Non competitive inhibition : Also reversible but not by
the substrate
It binds at a site on the enzyme other than the substrate
binding site, altering the conformation of the enzyme
molecule so that reversible inactivation of the catalytic
site results.
Noncompetitive inhibitors bind reversibly to both the
free enzyme and the ES complex to form the inactive
complexes EI and ESI
E +I EI, ES +I ESI
e.g. inhibition of L Threonine dehydratase by isoleucine