Enzymes

Contents of the course:

General concepts
What are enzymes?
Properties
Nomenclature
Enzyme specificity:
Specificity to the type of reaction catalyzed
Specificty to substrate
How enzymes work
Cofactors and Coenzymes
Factors affecting enzymatic activity
Kinetics
Inhibition of enzyme activity
Allosteric enzymes
Regulation of enzyme activity
Clinical application of enzymology
GENERAL CONCEPTS
General concepts:
A catalyst increases the speed of a chemical reaction but
itself is not changed after completion of the process
Speed of a reaction is called rate or velocity (V): change
in amount of reactants or products per unit time
WHAT ARE ENZYMES?
 Enzymes: are biolgical catalysts- mostly are proteins but
some RNA molecules also are involved in catalysis
Substrate (S): is the molecule acted upon by the enzyme
to form product (P)
PROPERTIES OF ENZYMES
Properties of enzymes:
1. extremely efficient
2. increase the rate of the reaction by lowering the energy of
activation
3. do not change the equilibrium constant of the reaction
4. do not change as a result of catalysis
5. have an active site
6. show specificity to substrate and to the type of reaction
7. exhibit optical specificity
8. some enzymes require coenzymes for activity
9. regulatability: their activity is regulated
NOMENCLATURE
Nomenclature:

Trivial naming
By adding the suffix ase to the name of the
substrate e.g., lipases, amylases, proteases
By the type of reaction catalyzed e.g., alcohol
dehydrogenase
Exceptions include older names like trypsin, pepsin,
etc
Systematic
nomenclature
IUB names the enzymes depending on their
classification. The name is of two parts: 1st part name of
substrate(s). The 2nd part ending with ase indicating the
type of reaction catalyzed e.g., ATP: hexose 6-
phosphotransferase
Each enzyme has a code (EC) of 4 digits: 1st digit is for
the class, 2nd one for the subclass, 3rd digit for the sub-
subclass and the 4th one denotes the specific enzyme,
e.g., 2.7.1.1. EC is used for research purposes.
 Enzyme specificity:
1. Specificity to the type of reaction catalyzed:
IUBMB classified all enzymes into six major classes, each
subdivided into subclasses that are further subdivided
Class 1:Oxidoreductases
Catalyze oxidation-reduction reactions
e.g., alcohol: NAD+ oxidoreductase (alcohol dehydrogenase)
Subclasses:
1. Dehydrogenases: remove two electrons and
two H atoms from the substrate
2. Oxidases: transfer two electrons from the
donor to oxygen resulting usually in H2O2 e.g.
glucose oxidase. Cytochrome oxidase produces
H2O
3. Oxygenases: add oxygen into a substrate
4. Peroxidases: utilizes H2O2 rather than oxygen
as the oxidant e.g., NADH peroxidase catalyzes
the reaction
NADH + H+ + H2O2 NAD+ + 2H2O
5. Catalase: H2O2 acts as donor and acceptor.
Catalase functions in the cell to detoxify H2O2
H2O2 + H2O2 O2 + 2H2O
 Dehydrogenases:

Oxygenases:

Oxidoreductases
Peroxidases:

Catalase

oxidases
Class 2:Transferases
These enzymes transfer functional groups between
substrates
e.g., for subclasses: phosphotransferases (kinases)
transfer phosphate groups like glucokinase or
hexokinase
glucose + ATP glucose 6-phosphate
Aminotransferases transfer amino groups
Class 3:Hydrolases
Enzymes which catalyze hydrolytic cleavage e.g., peptidases
Class 4: Lyases
Add or remove the elements of water, ammonia, or CO2 e.g.,
dehydratases remove H2O in a dehydration reaction
Class 5: Isomerases
Catalyze isomerizations of different types
Sublasses: epimerases, racemases, isomerases,
mutases

Class 6: Ligases
Catalyze joining of two molecules using energy (ATP).
They are called synthetases since they are involved in
synthetic reactions
2.Specificity to substrate:
Is explained by the active site and the binding site
The tertiary structure of an enzyme creates a region
on the enzyme surface that has the correct
molecular dimensions to accommodate a specific
substrate, i.e., there is complementarity in shape
between the active site and the specific substrate
The enzyme has affinity for its substrate
The active site is a particular arrangement of amino
acid side chains involved in catalyzing the reaction
There are levels of specificity:
Absolute specificity: the enzyme is specific to only one
substrate, like glucokinase
Broad specificity: the enzyme is specific to a few number of
substrates which are structurally related, like hexokinase
(glucose, fructose, mannose with different Km values)
 There are two models to explain the specificity of the
enzyme to its substrate:
A. Lock and key model:
The active site is preshaped
This model gives a rigid picture of the enzyme and
cannot account for effects of allosteric ligands.

B. Induced-fit model:
A more flexible model in which the binding and
active sites are not fully preformed
Interaction of the substrate and the enzyme induces
a conformational change in the enzyme resulting in
the formation of stronger binding site
 How enzymes work:
A simple enzymatic reaction might be written as
E +S ES EP E+P
There is an energy barrier between S and P that
represents the energy required for alignment of
reacting groups
Enzymes enhance reaction rates by lowering the
activation energy
Activation energy is the difference between the
energy levels of the ground state and the transition
state
 Energy to lower the activation energy is provided by catalytic
functional groups at the active site through the formation of weak
interactions between the enzyme and the substrate, i.e. formation
of ES complex is followed by a small release of free energy that
provides a degree of stability to the interaction
Cofactors & Coenzymes

Cofactors are small organic or inorganic molecules that are

required for the enzyme activity

Apoenzyme is the protein part of an enzyme without any

cofactor. It is catalytically inactive

Not all enzymes require cofactors (that require include classes

1, 2, 5 and 6)

Examples of inorganic cofactor molecules are metal ions e.g.,

Fe, Mg
 Organic cofactors are termed coenzymes

Coenzymes are small organic molecules often

derivatives of vitamins that function with the enzyme
in the catalytic process

Coenzymes can be considered as second substrates

because they have affinity for the enzyme and
chemical changes taking place in coenzymes exactly
counterbalance those taking place in the substrate
SH2 + NAD+ S + NADH + H+
 A prosthetic group is a cofactor that is tightly bound to an
apoenzyme
Apoenzyme + cofactor = holoenzyme (active form)
Examples of coenzymes are NAD+, NADP+, FMN, FAD,
Coenzyme A ( CoA )
 Coenzymes are classified into two groups:
Coenzymes for hydrogen transfer: NAD+, NADP+, FAD, FMN,
CoQ and lipoic acid
Coenzymes for transfer of groups other than hydrogen:
CoASH, thiamine pyrophosphate, folate derivatives, biotin,
B12 coenzymes and lipoic acid
Factors affecting enzyme activity
1. Temprature
2.pH
3.Enzyme concentration
4.Substrate concentraction
Factors affecting enzyme activity
1. Temp
Plot of velocity (V) or activity versus temp give a bell-
shaped curve for most enzymes
At first increase of V is due to the increased number
of molecules having sufficient energy to pass the
over the energy barrier and form the products of the
reaction
This is until a maximum velocity (Vmax) is reached
 Optimum temp is the temp that gives Vmax
Q10 is a factor by which the velocity increases for every 10C rise
Decrease of velocity with further elevation of temp causes temp-
induced denaturation of the enzyme
2.pH
Nearly all enzymes show a bell-shaped profile
Effect of pH is on the ionization of the amino acid
side chains involved in catalysis (active site) and in
the structure of the catalytically active enzyme
pH also affects the ionization of the substrate
Optimum pH is the pH that gives Vmax
Optimum pH varies with different enzymes e.g.,
pepsin has an optimum pH of about 2, whereas most
enzymes are designed to work at neutral pH
3.Enzyme concentration
The initial velocity of enzyme-catalyzed reaction is always
proportionate to the concentration of enzyme
4.Substrate concentraction
A plot of initial velocity Vo versus [S] gives a hyperbolic curve
The initial velocity of an enzyme catalyzed reaction is dependent on
the concentration of the substrate [S]
As [S] increases initial velocity increases until a maximum velocity
(Vmax). At this point the enzyme is completely saturated with
substrate ( all the active sites of the enzyme are occupied with the
substrate)
 The curve is referred to as the substrate saturation curve of an
enzyme-catalyzed reaction
Vmax is the velocity obtained under conditions of substrate
saturation of the enzyme
Kinetics (with reference to the previous figure)
The substrate saturation curve of an enzyme-
catalyzed reaction is defined by an equation known
as Michaelis-Menten equation

Vo = Vmax[S] Km +[S]
Km (Michaelis constant) and Vmax are unique to
each enzyme under specific conditions of pH and
temp.
Vmax does not depend on the substrate
concentration [S] , unit: mol/L / s
 Km corresponds to the substrate concentration at which V is
half Vmax (when v = 1/2 Vmax). KM has a unit mol/L = M.
Most enzymes obey Michaelis-Menten kinetics; exceptions are
allosteric enzymes (regulatory enzymes)
Significance of Km
Km equals the substrate concentration that gives half the maximum
velocity
it is reciprocally related to the affinity of the enzyme to its substrate
Km can vary greatly from enzyme to enzyme and even for different
substrates of the same enzyme
 Km has a physiologic significance e.g., hexokinase (in all
tissues) has a lower Km compaired to glucokinase ( in liver and
pancreas)
Lineweaver-Burk or double-reciprocal plot
Its the linear form of Michaelis-Menten equation
It is used to determine Km values by taking the reciprocal of Vo
versus the reciprocal of the [S]. This gives a straight line
1 Vo = Km Vmax 1 [S] +1 Vmax
This equation is useful in analyzing enzyme inhibition
Lineweaver-Burk or double-reciprocal plot
Inhibition of enzyme activity
Any substance that can diminish the velocity of an enzyme-
catalyzed reaction is called an inhibitor
Inhibition of enzymatic activity by specific small molecules and ions
is important because it serves as a major control mechanism in
biologic systems. Also many drugs act by inhibiting enzymes
There are two classes of inhibition:
Reversible: the inhibitor binds the enzyme through noncovalent
forces .
Irreversible: the inhibitor binds tightly, either covalently or
noncovalently, to the enzyme
A. Reversible inhibition
1. Competitive inhibition
The competitive inhibitor is structurally similar to the
substrate
Therefore it binds the active site and lowers the
affinity of the enzyme to the substrate ( increases
Km)
Vmax does not change
Competitive inhibition can be overcome by
increasing the substrate concentration
e.g., malonate competitively inhibits succinate
dehydrogenase
2. Noncompetitive inhibition
The noncompetitive inhibitor has no resemblance to
the substrate
Therefore it binds at a site other than the active site
Inhibition is not reversed by increasing the substrate
concentration
It binds to E or ES
A noncompetitive inhibitor decreases Vmax , but
does not change Km
Noncompetitive inhibition can be reversed by dialysis
3.Uncompetitive inhibition
The uncompetitive inhibitor binds only with the ES form
Results in equivalent change in Vmax and Km
A. Irreversible inhibition
An irreversible inhibitor inactives enzymes by forming strong
bonds to a particular group at the active site.
Is not reversed by dialysis or by addition of substrate
e.g., lead poisoning. Lead form covalent bonds with SH side
chains of cysteine
The nerve gases irreversibly inhibit biological system by
forming an enzyme-inhibitor complex with a specific hydroxyl
group (of serine) situated at the active site of certain
enzymes.
Hydrogen cyanide (HCN) inactivates iron-containing enzymes
(catalase, cytochrome oxidase).
Allosteric enzymes:
Allos means other, Steric means space
Allosteric enzymes are enzymes whose activity at
the catalytic site may be modulated by the presence
of allosteric effectors at an allosteric site
Most allosteric enzymes are oligomeric proteins
The small regulatory molecules to which they bind
are known as allosteric effocters
 Allosteric effectors have little or no structural
resemblance to the substrate, they cause a
conformational change in the enzyme so that the
affinity for the substrate or other ligands also change
When those effectors bind to enzymes, they alter the
catalytic properties of an enzymes active site. If the
effector causes an increase in enzyme activity it is
called positive allosteric effector, in case it lowers the
activity it is referred to as a negative allosteric
effector
 Allosteric effectors affect Km or Vmax or both
Thus allosteric enzymes are regulatory enzymes
catalyzing committed steps or rate limiting steps
Cooperativity is the infuence that the binding of a
ligand to one promoter has on the binding of a ligand
to another promoter in an oligomeric protein
Thus allosteric enzymes show cooperativity and
sigmoidal substrate saturation curve
They don't obey Michaelis-Menten kinetics
Regulation of enzyme activity:

The activity of the enzyme can be regulated in a number

ways:
1) Compartmentation:
distribution of the pathways in different organelles
2) Induction-repression and proteolytic degradation:
The absolute amount of the enzyme can be regulated by change in
the synthesis of the enzyme (in this case the enzyme is called
inducible enzyme e.g.,glucokinase) and/or by change in the rate of
degradation.
 Enzyme synthesis and proteolytic degradation are
comparatively slow mechanisms for regulating enzyme
concentration, with response times of hours, days or even
weeks. Its a long-term regulation (adaptation). Degradation of
enzymes is by proteolysis which is irreversible
3) Allosteric regulation:
The activity of the enzyme can be modulated by activators or
inhibitors through allosteric effects. Its a short-term
regulation, with response time of seconds. Its a reversible
type of regulation
4) Reversible covalent modification:
Regulation of the activity of the enzyme is through
covalent binding of a functional group.
The common type is by phosphate binding
Protein kinases bind the phosphate group to the
enzyme
Protein phsphatases remove the phosphate group
from the enzyme
Thus its a reversible type of regulation (reversed by
the activity of kinases & phosphatases)
5) Irreversible covalent modification:
Proenzymes or zymogens are inactive forms of enzymes and
are generally synthesized in abundance, stored in secretory
granules and covalently activated upon release from their
storage sites (by cleavage of specific peptide bonds)
Proenzyme activation is a more rapid method of increasing
enzyme activity but, as a regulatory mechanism, it has the
disadvantage of not being a reversible process
Examples of important zymogens include the digestive
enzymes pepsinogen, trypsinogen and chymotrypsinogen
Likewise, many of the blood clotting proteins are synthesized
as proenzymes