Enzymes

Proteins with catalytic function

Enzymes - biological catalysts in conditions:
– relatively low temperature (370C),
– pressure - 1 atm,
– specific pH (7.2 – 7.6)
– Net superior catalytic effect, decreasing activation energy of
reactants
– In the reversible reactions, they catalyze reactions in both
senses
⇒very efficient catalysts.
• RNA – ribozymes
Kinetic theory of collisions in chemical reactions
• reactants should have:
– correct geometric orientation,
– favorable for collision between reactants,
– enough energy to collide.
• high energy intermediate
• Activation energy - energy required by molecules found in 1 mol of
reactants to have effective collisions
• A reaction is appreciated by standard free energy in physiological
conditions (G0`).
– t = 250C
– p = 1 atm
– [reactant] = 1 mol/liter
– pH = 7

• The change in free energy (∆G) for the overall reaction is

independent of catalysis;
• the equilibrium constant for a chemical reaction is a function of the
standard free energy change:
∆G0` = - RT lnKeq

• ∆G0` is also function of the electric potential for redox reactions

∆G0` = - nF∆E`
∆G0` < 0 ⇒ spontaneous, or exergonic reactions ⇒ energy release
∆G0` > 0 ⇒ endergonic reaction - need energy to proceed
∆G0` = 0 ⇒ reaction at equilibrium

Catalytic comparative efficiency of chemical H2O2 decomposition

Relative Activation energy

Catalyst
reaction rate (kcal/mol)
Non catalyzed 1 18
Iodides 8 x 102 14

Platinum 2 x 104 12
Catalase 3 x 1011 5
 Characteristics of enzymes
Localization
• found in all body cells.
• abundance increases with the biochemical activity level
⇒ pancreatic and hepatic cells, more than 70% of proteins have
enzymatic activity.
• depends on the localization of metabolic pathways in cell.
⇒ each cell organelle has its own characteristic enzymes.

Examples
– plasma membrane: 5`-nucleosidase, acetylcholine esterase
– cytosol: LDH (lactate dehydrogenase)
– peroxisomes: catalase
– endoplasmatic reticulum (ER): glucoso-6-phosphatase
– mitochondria: succinate dehydrogenase
• In some cases, enzymes form multifunctional protein complexes
⇒ many successively catalytic actions
(e.g. pyruvate dehydrogenase).
 Characteristics of enzymes

Specificity

• Specificity of action or reaction specificity (e.g. phosphatases

hydrolyze ester phosphates)
• Specificity of substrate:
– Large specificity (e.g. phosphatases hydrolyze esters of
phosphoric acid with different alcohols)
– Group specificity (e.g. esterase hydrolyzes esters of the same
alcohol with different acids)
– Absolute specificity (e.g. urease hydrolyzes exclusively urea to
CO2 and NH3;
– Stereo-specificity: α-amylase hydrolyzes only α(1-4) bonds
from starch and/or glycogen
 Enzyme structure
• depends on the catalytic action of that enzyme.
• action could be done with or without contribution of some non
proteic components - enzymatic cofactors

Classification:
• Simple enzymes – consist only of protein part
• Complex enzymes (holoenzymes), which are composed of:
– apoenzyme - protein component - responsible of enzyme
substrate specificity
– enzymatic cofactor - non-proteic component - responsible of
enzyme action specificity.
 Enzyme structure
Enzymatic cofactors:
- coenzyme – small molecular mass organic compounds.
- derivatives of vitamins:
- pyridoxal phosphate (B1),
- FAD and FMN (B2),
- NAD+ and NADP+ (B3, niacine),
- nucleoside triphosphates
- ATP, UTP, CTP, GTP
- prosthetic group
- the small organic molecule is bound to the
apoenzyme by covalent bonds.
- It cannot be detached from enzyme.
 Enzyme structure
• Functional role of coenzymes: act as transporters of chemical groups
from one reactant to another.
– hydride ion (H+ + 2e-), carried by NAD+,
– or hydrogen molecule carried by FAD;
– more complex like amine (-NH2) carried by pyridoxal phosphate.
• coenzymes are chemically changed as a consequence of enzyme action, it
is often useful to consider coenzymes to be a special class of substrates, or
second substrates, which are common to many different holoenzymes
Examples:
Glucose + Hexokinase–ATP → glucose-6-P + hexokinase–ADP
Pyruvic acid + lactate dehydrogenase–NADH(H+) → lactic acid +
lactate dehidrogenase–NAD+

Succinic acid + succinate dehidrogenase–FAD → fumaric acid +

succinate dehidrogenase–FADH2
 Enzyme structure
• metalloenzymes - bind and retain their metal atom(s) in all
conditions
• metal-activated enzymes - have a lower affinity for metal ion, but
still require the metal ion for their activity
• Binding of metal ion is function of apoenzyme, therefore enzymes
that catalyze the same type of reaction can have, function of
tissue, different metallic cofactors.

Role of metal:
– Participate in the binding of substrates and enzyme cofactors:
Mg2+ - ATP.
– Stabilize the active conformation of the enzyme.
– Act as Lewis acid in electrophile catalysis.
– Acts electron donor substrate in oxidation-reduction reactions
(Fe, Zn, Cu, Mn, eg Cu2+ ⇔ Cu+).
Cu and Fe may have role in oxygen activation in the structure of
some oxidases and hydrolase
 Enzyme action
• possess specific sites on their surface, for substrate
binding.
• Enzyme conformation creates complementary regions
(size, topology, electrical charge) with the substrate
• Tolerance of these sites may be so small that only one
isomer in a pair of diastereoisomers will be bound.
• For example, only D-amino acids will be oxidized in the
catalysis of D-aminoacid oxidase.

Models of action:

• lock and key model (Fischer, 1895)

• induced fit model (Koshland, 1968)
Lock and key model assumes a high degree of similarity between the
shape of the substrate and the geometry of the binding site on the
enzyme. The substrate binds to a site into which it fits exactly, like a
key in a lock or a right piece in a three-dimensional jigsaw puzzle.

• explained enzyme specificity

• not all enzymes have the specific shapes required for the lock-and-
key theory.
• This rigid model cannot explain the catalytic act and the allosteric
effects.
Induced fit model considers the existence of substrate binding sites
and active, catalytic site that transform the substrate.
• These sites have no a performant orientation, but interaction of
enzyme with substrate induces a modification of enzyme
conformation, followed by a “harmonization” with substrate structure.
• This harmonization involves both the binding sites and the active,
catalytic sites, which form the catalytic center.
• Conformational modifications produce catalytic actions (acid-base,
electrostatic, covalent) upon substrate bonds, after which they break
bonds in the substrate molecule, forming new bonds and molecules.
Catalytic active center of an enzyme:
• the delimited area of enzyme that achieves the substrate
binding and transformation
• It contains binding sites, which orients specifically the
substrate in order to obtain optimum position for
achieving the catalytic act, and catalytic active sites,
which brake up bonds in substrate molecule and forms
new ones.
– In binding sites, non-polar amino acids predominate (Ala, Val,
Leu, Phe), which will form hydrophobic bonds with the substrate
– In active, catalytic sites, polar or ionisable amino acids
predominate (Ser, Cys, Glu, Asp, Lys, Hys). In general, their
number is very small, 2-5 amino acid residues. For example, in
case of ribonuclease, of 124 amino acid residues, only 2 of
them, Hys12 and Hys129 are part of the active catalytic center.
Action to reduce entropy
• In a diluted solution, two reactants have different
orientations, fact that reduces the chance of a correct
geometric oriented collision and of low activation energy
at 37°C.
• Introduction of an enzyme with binding sites for the two
reactants, will force them to attach to the enzyme into an
orientation and stereochimiometry propitious to reaction.
• Correct orientation and approach of reactants will reduce
entropy, this factor being considered responsible for the
increase of 103 times of the speed of reaction.
 Mechanism of enzyme action

• Catalytic action takes place in the catalytic active center consisting

of 3-5 amino acid residues.
• Several catalytic mechanisms - function of the nature of amino
acid residues in the active center, which participate to the formation
of reaction intermediary (ES)
• it is necessary reactants have sufficient energy to overcome the
activation energy barrier. Reactants found in this posture constitute
a complex called transition state (Ts). This complex may evolve
into products or back to the reactant state.
• enzyme permits the fragmentation of activation energy.
• enzyme has a much higher affinity for the transition complex Ts.
Thus, the fraction of substrate molecules that exists as Ts form will
be rapidly transformed into products and then, following the low of
chemical equilibrium, the whole quantity of substrate will be rapidly
converted into products.
 CATALYTIC STRATEGIES

• ACID-BASE CATALYSIS
• COVALENT CATALYSIS
• CATALYSIS BY STABILISATION OF THE
TRANSITION STATE
 CATALYTIC STRATEGIES
1. Acid - Base Catalysis
• the acidic or basic groups (other than water)
could serve as proton transfer agents.
• At the physiological pH, the protonated form of
His is the most important acid and its
conjugated base an important base also (other
acids: HS-Cys, HO-Tyr, ε-amino-Lys and other
bases: their conjugated bases).
Example 1: the mechanism of action of trioso-
phosphate isomerase, enzyme that catalyzes
the reaction (chapter 16.1):
O OH
-2 -2
HO OPO3 O OPO3

Dihydroxyacetone phosphate (DHAP)→

glyceraldehyde 3-phosphate (GA3P)
Exemple 2. Chymotrypsine, a digestive enzyme uses a
Hys residue as a base catalyst to enhance the
nucleophilic power of serine.
THE CATALYTIC TRIAD

Asp -Hys-Ser
2. Covalent Catalysis:

• the substrate is oriented to active sites on the enzymes

in such a way that a covalent intermediate forms
between the enzyme or coenzyme and the substrate.
The active site of the enzyme contains a reactive group ,
usually a powerful nucleophile, that becomes temporarily
covalently attached to the substrate in course of
catalysis
– a. Electrophile catalysis:
• lateral chains can stabilize the intermediates with
opposite charge (e.g. carboxylate anion of Glu,
Asp residues from active site can stabilize a
carbocation).
• A positive metallic ion from the catalytic site can
stabilize an oxyanion
• ex. SUPEROXIDE DISMUTASE
2H2O + O●-2→ 2H2O2
– b. Nucleophile catalysis:
• a nucleophilic agent from enzyme (e.g. OH- group
of serine) attacks a group from substrate by
nucleophilic attack and forms an intermediate,
more reactive than the initial substrate.
• This is the mechanism of serine proteases
action (have a common mechanism of action on
substrate, but they have different specificity:
– Elastase hydrolyzes peptidic bonds inside the
protein at carboxyl group of Gly and Ala
residues (e.g. in fibrous proteins as elastin)
– Trypsin hydrolyzes peptidic bonds inside the
protein at carboxyl group of Lys and Arg
residues
– Chymotrypsin hydrolyzes peptidic bonds
inside the protein at carboxyl group of
aromatic amino acid residues such as Tyr,
Phe, Trp
Exemple1: Chymotrypsin
Catalyze the protein hydrolysis
by nucleophilic attack to a
carbonyl C atom of an
aromatic AA or large
hydrophobic AA
• 3 clases of peptide bond hydrolysis

Cysteine proteases
Aspartyl proteases
Metalloproteases

The strategy is to generate a nucleophil that attacks the

carbonyl peptide bond
Exemple2:
Carbonic anhydrase
Catalyze the hydration of CO2
• The binding a water molecule to the Zinc ion of carbonic
anhydrase favors the formation of the transition state by
facilitating the proton release and the positioning of
water molecule to be in close proximity with the other
reactant (CO2)
Exemple 3. DNA cleavage by EcoRV endonuclease action

Restriction enzymes requires Mg2+

3. Catalysis by stabilization of transition state of
reactants
• In the first step reactants are transformed by the active
center of the enzyme into a transition state (reaction
intermediate), which later will become the final product of
reaction.
• Since the active site binds the transition state
compounds with much higher affinity than the substrate,
the intermediate formation is favoured.
• Transition state is stabilized by:
– better sterical fitting between the active site of the enzyme and
the transition intermediate than between the active site of the
enzyme and the substrate
– The polar rests from the active site bind the transition state
which possesses partial charges and, also, the lipophilic
residues stabilizes the intermediate.
• An example of this mechanism is the catalytic action of the
adenyl-deaminase:

NH2 OH
H2O H2N OH
NH3 N
N N N
N HN

N N
N N N N
Riboză
Ribose
Riboză
Ribose Riboză
Ribose
Adenozinã Intermediar
Intermediate Inozinã
Inosine
Adenosine

• Another example is that of Myosin mediated ATP hydrolysis

 Isozymes

• different enzymes have a more or less broad range of substrate

specificity
• a given substrate may be acted on by a number of enzymes, each of
which uses the same substrate(s) and produces the same
product(s).
• The individual members of a set of enzymes sharing such
characteristics are known as isozymes.

Isoenzymes have different physicochemical properties:

• thermostability
• optimal pH under which they may be separated
• electrophoretic migration
• Michaelis constant
 Isozymes
Differentiation of isoenzymes

• It results by genetic differences between protein molecules. One can

distinguish three categories:
– Isoenzymes whose genes are totally different. Example:
malate dehydrogenase in cytoplasm and mitochondrion are
enzymes with primary structure completely different.
– Isoenzymes heteropolymer type, formed by different
combinations of two or more polypeptide chains. Example:
LDH – lactate dehydrogenase (M = 132000) is formed of four
chains of two types α and β, resulting 5 isoenzymes
– Aleloenzymes which consist of proteins that differ by a
punctual unit (replacing of one amino acid with another).
 Isozymes
Isoenzymes with clinical applications

• Creatinkinase (CPK) is a dimeric enzyme formed of two types of

subunits: M (muscle type) and B (brain type). Three isoforms can be
distinguished, with structure:
– MM (skeletal muscle) CPK3
– MB (only in myocardium) CPK2
– BB (in brain) CPK1
• Isoenzymes are numbered in order of migration to anode.
• Lactate dehydrogenase – is a tetrameric enzyme, formed of two
types of subunits: H (myocardium) and M (muscle). Five isoforms
can be distinguished, with structure
– LDH1 HHHH myocardium and RBC
– LDH2 HHHM myocardium and RBC
– LDH3 HHMM brain and kidney
– LDH4 HMMM -
– LDH5 MMMM liver and muscle
After a myocardial infarction:
• serum CPK2 increases at 6 -18 hours after infarction
• LDH1 and LDH2, having been a reversal of ratio from LDH2 > LDH1
to LDH1 > LDH2.
• These findings are diagnosis of myocardial infarction in 100% of
cases.

Increase of LDH5 activity is associated with a liver congestion.

 Kinetic of enzymatic reactions
Enzymatic kinetic- terminology
• velocity - is measured in µmoles of substrate transformed in
products per minute at 25°C, 1 atm and the optimum pH of the
enzyme.
• enzymatic activity - represents µmoles of substrate transformed in
products per minute at 25°C, 1 atm and optimum pH of the enzyme
by the volume of enzyme (L) or by the enzyme quantity.
• international standard enzyme activity unit - quantity of enzyme
that transforms 1 µmole of substrate per minute.
• specific activity - depends on the number of enzymatic units per
mg of protein (µmol min-1 / mg protein)
• Catalytic constant (Katal) - number of activity units per mole of
enzyme. Newly, the Katal (Kat) = 1 mol substrate / 1 sec.
• Maximal velocity (Vmax) - obtained in conditions of enzyme
saturation with substrate and optimum conditions of pH,
temperature, ions concentration. Vmax is a constant, specific to
each enzyme.
 General characteristics of catalyzed reactions

• reactant and product concentrations are usually

hundreds or thousands of times greater than the enzyme
concentration.

⇒ each enzyme molecule catalyzes the conversion to

product of many reactant molecules.

• In biochemical reactions, reactants are commonly known

as substrates

• enzyme saturation with substrate: reaction velocity

increases with substrate concentration up to a certain
value (when all enzyme catalytic sites are occupied) and
then the reaction velocity remains constant.
• In case of enzymatic reactions, three stages may be distinguished:
– Substrate fixation [S] on enzyme [E] complex formation of [ES]
– Transformation of bound substrate [ES] into bound product [EP]
– Complex dissociation [EP] into [E] and [P].
k +1 k +2 k +3
E + S ES EP E + P
k -1 k -2 k -3

– At the beginning of the reaction there is no P and one can say

that complex [EP] dissociates faster than EP is formed from E
and P, so k3 > k2.
⇒ reaction that limits the speed of P formation is the second
one, characterized by k+2.

k +1 k +2
E + S ES E + P
k -1
 k +1 k +2
E + S ES E + P
k -1

• Leonor Michaelis and Maud Menten developed an equation that

describes the velocity of product formation depending on substrate
concentration. d [P]
v0 = = k + 2 [ ES ]
dt

• ES synthesis: v f = k 1 [E] [S]

• ES dissociates: v d = (k -1 + k + 2 ) [ES]

• the theory of „steady state” (stationary state), in any moment of

the enzymatic reaction, the velocities of formation and breakdown of
ES are equal and [ES] = constant
 v 0 = k + 2 [ES]

The velocity is maximal when the enzyme is free and able to bind the substrate
⇒
Vmax = k + 2 [E t ]

k + 2 [Et] [S] Vmax [S]

v0 = = Michaelis-Menten equation
K M + [S] K M + [S]

- KM is called the Michaelis constant.

- KM has units of concentration and, because it is a ratio of the rate
constants of a reaction, KM is characteristic of the reaction.
- A given enzyme acting upon a given substrate has a distinct KM.
 vd = vf

(k -1 + k 2 ) [ES] = k 1 [E] [S]

k +1 [E] [S]
[ES] = [E] [S] =
k -1 k + 2 KM

[E] = [E] tot - [ES]

([Et] − [ES]) [S] [Et] [S] [ES] [S]

[ES] = = - [E] - free enzyme;
KM KM KM
[Et] or [E]tot - total enzyme.

[S] [Et] [S]

[ES](1 + )= x KM
KM KM

[ES](K M + [S]) = [E t ] [S]

E t [S]
[ES] =
K M + [S]
Graph representation: rectangular hyperbola
For special cases:
• [S] >> KM v0 = Vmax
reaction rate independent of
substrate concentration [S]
• [S] = KM v0 = Vmax/2
hemisaturated enzyme
• [S] << KM v0 = [S]Vmax / 2
reaction rate is directly
proportional to substrate
concentration [S]
[S] << KM [S] = KM [S] >> KM
KM significance
k +1 k +2
E + S ES E + P k + k +2
k -1 KM = -1

k +1

- apparent dissociation constant v

and substrate concentration at
which the enzyme is half saturated
Vmax
and the reaction velocity is is half
of maximal velocity
- KM value is an indicator of Vmax S3
S1 S2
enzyme affinity for its substrate 2

- KM is inversely proportional to the

intensity of affinity
- In general, enzyme substrate 1 23 [S]
affinity has values:
10-5M < KM < 10-2M.
• To avoid dealing with curvilinear plots of enzyme catalyzed
reactions, biochemists Lineweaver and Burk introduced an
analysis of enzyme kinetics based on the following rearrangement of
the Michaelis-Menten equation:
 Mechanism of enzyme reactions
with two substrates

• Most enzymes catalyze reactions between two or more

substrates, yielding one or more products.
• For some enzymes, all substrates must be present
simultaneously for the reaction to occur.
• For others, the enzyme first alters one substrate and
then catalyzes its reaction with the second substrate.
• the kinetic is more complicate,
• As mechanism, there are two categories:
– 1. Sequential mechanism
• The ordered sequential mechanism
• The random sequential mechanism
– 2. The ping-pong (double displacement) mechanism
• 1. Sequential mechanism – the substrates are fixed at the active
center before the products to be liberated.
– a) The ordered sequential mechanism
• the substrates bind to the enzyme, and the products are released in
a defined sequence. Firstly the two substrates bind to give an
enzyme substrate complex:
E + A ↔ EA
EA + B ↔ (EAB)
• The enzyme substrate complex is now converted to enzyme product
complex:
(EAB) ↔ (EPQ)
• The products are now released:
(EPQ) ↔ EQ + P
EQ ↔ E + Q

Cleland plot
• 1. Sequential mechanism
– b) The random sequential mechanism
– there is no specified order in which substrates must bind or products be
released. Initially the substrates bind, but they can do so in either order:
E + A ↔ EA or E + B ↔ EB
EA + B ↔ (EAB) or EB + A ↔ (EAB)

– central complex is identical in both cases.

– The next step is the same as with an ordered mechanism:
(EAB) ↔ (EPQ)
– We now have a choice of sequences of product release:
(EPQ) ↔ EQ + P or (EPQ) ↔ EP + Q
EQ ↔ E + Q or EP ↔ E + P

Cleland plot
• 2. The ping-pong (double displacement) mechanism
• It is a mechanism characteristic to transfer reactions of chemical groups
from a substrate to another, by the enzyme. The process includes two
reactions:
• Binding of A and its transformation to P
• ⇒ the enzyme is modified by attaching chemical group assigned to A.
• B binding, which has affinity for the modified enzyme and conversion to Q
• ⇒ the enzyme back to its original state unchanged.

•E + A ↔ (EA)
•(EA) ↔ (FP)
•(FP) ↔ F + P
•F + B ↔ (FB)
•(FB) ↔ (EQ)
•(EQ) ↔ E + Q
Cleland plot

An example is aminotransferases, enzymes that contain

pyridoxal-phosphate as prosthetic group and transfer an
amino group from an amino acid (A) to a ketoacid (B).
• Enzyme-pyridoxal + glutamic acid →
Enzyme-pyridoxamine + α-ketoglutarate

• Enzime-pyridoxamine + pyruvic acid →

Enzyme-pyridoxal + Alanine

α - cetoglutarat
CH2 NH2 CH2 NH2
CHO CHO
Glu E E
E E α - cetoglutarat
Glu

CH2 NH2 CHO CHO

CH2 NH2
Piruvat E + Alaninã
E E E
Piruvat Alanina
 Factors involved in enzymatic kinetic
Temperature

• Every enzyme has a temperature

range of optimum activity.
• Outside that temperature range the
enzyme is rendered inactive and is
said to be totally inhibited.
• as the temperature changes this
supplies enough energy to break
some of the intramolecular
attractions between polar groups
(hydrogen bonding, dipole-dipole
attractions) as well as the hydrophobic
forces between non-polar groups
within the protein structure.
• When these forces are disturbed and Most enzymes in the body have an
changed, this causes a change in the
secondary and tertiary levels of optimum activity around 37°C,
protein structure, and the active site temperature increase over this value
is altered in its conformation resulting in loss of enzymatic activity.
beyond its ability to accommodate the
substrate molecules it was intended to
catalyze.
 pH

• Changes in the pH or acidity

would alter or totally inhibit the
enzyme from catalyzing a
reaction.
• will affect the polar and non-
polar intramolecular attractive
and repulsive forces and alter
the shape of the enzyme and
the active site as well to the
point where the substrate
molecule could no longer fit,
and the chemical change
would be inhibited from taking
place as efficiently or not at all.
• Every enzyme has an
optimum pH range outside of
which the enzyme is inhibited.
Enzymatic activators

• Stimulate enzyme activity ⇒ activators

1. Direct activation

• Ion activation: Mg2+, Mn2+, Zn2+, Co3+ and anions like Cl- have
action on some enzymes (e.g. Cl- is indispensable for salivary
amylase activity, specially to reach the maximal activity).
• Activation by unmasking the enzyme:
– Synthesised as proenzymes or zymogenes.
– Conversion to active enzyme involves selective proteolysis.
• Examples:
– hormone insulin,
– digestive enzymes pepsin, trypsin and chymotrypsin
(proproteins = pepsinogen, trypsinogen, and chymotrypsinogen,
respectively)
– several factors of the blood clotting and the blood clot
dissolution cascades,
– connective tissue protein collagen (proprotein = procollagen).
2. Indirect Activation

– It is, in fact, an anti-inhibiting action

– it is realized by removing or neutralizing of some inhibitors.

• For example:
– potassium cyanide (KCN) (a very strong inhibitor of Fe3+
enzymes) is an anti-inhibitor for urease: copper ions, which
inhibit urease, form a complex with cyanide, which has no
inhibiting effect.
– Also, –SH groups in the active center of some enzymes are very
sensitive to oxidizing agents. Vitamin C, glutathione, cysteine,
reducing compounds, protect –SH groups, annihilating the
oxidizing effects.
Enzymatic inhibitors

– factors that reduce or annihilate enzyme catalytic activity.

– Inhibition can be reversible or irreversible.

• Inhibitors may be classified as:

- Reversible inhibitors:
• Competitive
• Non-competitive
• Uncompetitive
– Irreversible inhibitors
– Antienzymes
1. Competitive inhibitors
– Are structural analogs of
substrate
– Bind to active center as
substrate
– Alter (decrease) enzyme
affinity for substrate, altering
the rate of catalysis
– Do not affect maximal velocity
of the enzyme
– Inhibition depends on
inhibitor/substrate ratio 1
V
Inhibited reaction
reacţie inhibată

E + S ES [S]>[I]
Non-Inhibited
reacţie neinhibată
reaction

E + I EI [I]>[S] 1
Vmax
1
1 1 [S]
EI + S ⇔ ES + I KM K'M
Bacteria synthesize folic acid from PABA in an enzyme
catalyzed reaction. They need the folic acid to make cell
walls.
Humans also need folic acid, but are incapable of
synthesizing it. It is a vitamin, which must come form
diet
2. Noncompetitive inhibition
• molecule or ion binds to a site on the
enzyme other than the active site
(allosteric)
• modifies the configuration of active
center.
• need not to resemble the substrate at
all.
• it needs to have a strong affinity for
the second binding site
• Inhibition depends on inhibitor
concentration
• KM remains unmodified (same affinity)
• Vmax decreases reacţie inhibată
1 Inhibited reaction
V
E + I → EI reacţie neinhibată
Non-Inhibited
reaction
1
V'max

1
Vmax 1
1 [S]
KM
3. Uncompetitive inhibition
• In this case inhibitor binds substrate-
enzyme complex.
• these inhibitors should change the
apparent values of Km as well as
Vmax.
• KM increases (affinity decreases)
• Vmax decreases

k+1 k+2
E + S ES E + P
k-1
kIS kIS

ESI
4. Irreversible inhibitors
• Are substances which are fixed to the enzyme through irreversible
covalent bond.
• lead to blocking enzyme activity.
• can be connected either to the active center or elsewhere.
Examples.
– Iodoacetates and mercury compounds react with SH groups of
free cysteine residues in the catalytic active center,
– diethylpirocarbonate reacts with histidine in the active center.
– A particular case is the metaloenzymes - EDTA binds metal ion
leading to inhibition or blocking of the enzyme.

5. Inhibition by antienzymes
• Antienzymes are proteic substances
• For example, in blood there are permanently inhibitors as α1-
antitripsin or α1-antitrombin, which neutralize the respective
enzymes.
• Intestinal parasites, like Ascaris lumbricoides, secrete inhibitors of
trypsin and chimotrypsin that explains the survival of these parasites
in the intestine.
 Therapeutic applications of
enzyme inhibitors
Inhibitor Target enzyme Action

acetylsalicylic acid (Aspirin) Cyclooxigenase Anti-inflammatory

Penicillin Bacteria Transpeptidase Antimicrobial

β-aminopropionitril Lysine oxidase Collagen diseases

Captopril (Lopril) Convertion enzyme of angiotensin in AHT (Arterial hypertension)

Atorvastatin (Tahor) HMG-CoA reductase In atherosclerosis

Allopurinol (Zycloric) Xantin oxidase in gout

GMP-phosphodiesterase
Sildenafil (Viagra) erecting trouble
type 5

Monoamin oxidase
Selegilin In Parkinson
type B

Finasterine (Chibro-proscar) Testosterone 5 α-reductase benign prostate hyperplasia

Omeprazol (Mopral) H+, K+ ATP-ase gastro duodenal ulcer

Ibuprofen Prostaglandin syntethase inflammatory processes

Methotrexat Dihydrofolate reductase Cancer

Orlistat (Xenical) gastrointestinal lipase Obesity

 Regulation of enzyme activity

• Changing environmental conditions require continuous adaptation

response of the body by altering the metabolic response.
• Since metabolic pathways are enzyme catalyzed reaction
sequences, regulation of enzyme activity is the main element in
metabolic changes.

• Setting the action of enzymes is achieved through two main

mechanisms:
• Change the amount of enzyme
• Change of enzyme activity by:
» allosteric changes (inhibitors, activators) or
» covalent modification.
1. Regulation of the amount of enzyme

– Is a primitive mechanism, slow, consuming energy and matter.

– The mechanism involves regulation of:
• Enzyme synthesis process (transcription, traslation)
• Enzyme catabolism
• It may be:
• Induction of enzyme synthesis: substrate functions
as an inducer in bacteria and in mammalian - hormonal
signals.
• represion of enzymatic synthesis: the reaction
product functions as repressor in bacteria and mammalian
- hormonal signals.
• degradation of enzymes
2. Regulation of enzyme activity

• do not affect enzyme concentration,

• are reversible and rapid in action,
• actually carry out most of the moment-to-moment physiological
regulation of enzyme activity.

• These mechanisms include:

– allosteric regulation,
– regulation by reversible covalent modification and
– regulation by control proteins such as calmodulin.
A. Allosteric regulation
• enzymes bind small, physiologically important molecules and
modulate activity
⇒ allosteric enzymes;
⇒ effectors (positive – activators; negative – inhibitors)
• Allosteric effectors
– bind to the enzyme at distinct allosteric sites, distant from the
catalytic site,
– cause conformational changes that are transmitted through the
bulk of the protein to the catalytically active site(s).
• Example: allosteric regulation of a metabolic pathway through its
product, which inhibits its own synthesis, inhibiting the enzyme that
catalyses the limitative step of this metabolic pathway:
⇒ Negative feed-back
E

S1 S2 S3 ...... Sn P
• From the kinetic point of view, there are two classes of enzymes that
suffer allosteric regulation:
– Enzymes of class K – the allosteric effector affects amino acid
residues from binding sites, modifying KM. Vmax remains at the
same value (unmodified).
– Enzymes of class V – modifications in catalytic sites affect
Vmax value, but KM remains unmodified.

• Allosteric effect, for oligomeric enzymes that are composed of a

single type of monomer, is of two types:
– Homotropic – binding of the effector to one of the monomeric
units influences the binding of the same ligand to the rest of
monomers.
– Heterotropic – binding of an effector influences the binding of
other effector of different type.
• The influence of an effector binding
to one promoter upon the binding to
the rest of promoter is called
cooperativity.
• It produces a sigmoid curve of
reaction rate over substrate
concentration.
• In case of oligomeric enzymes with
two types of monomers, these may
be:
• Regulatory subunits that have
allosteric sites for positive effectors,
negative effectors and binding sites
for catalytic subunits
• Catalytic subunits
For example: proteinkinase A is a
tetramer formed of two types of
promoters, 2R and 2C.
Binding of the positive effector,
cAMP, to regulatory subunits (R)
liberates the catalytic subunits (C)
from the inactive tetramer, and the
enzyme becomes active
B. Covalent regulation
• Is achieved by covalent attachment or detachment of a group to the
enzyme molecule.
• The most common way - regulation by phosphorylation -
dephosphorylation,
• the group attached - detached forms phosphate ester links with OH
group of Ser, Tre, Tyr residues in enzyme constitution.
• Phosphate donor is ATP
• enzymes that performs the process are:
• Kinases for attaching the phosphate group
• phosphatase for hydrolysis of phosphate group

Kinase

phosphatase
• Enzymes regulated by phosphorylation - dephosphorylation can be
active:
– in dephosphorylated form (glycogen synthase, pyruvate
dehydrogenase) or
– In phosphorylated form (phosphorylase, triglyceride lipase).

• Other reversible changes by covalent attachment may consist of

attachment – detachment of:
– AMP residue (adenylation) or
– ADP (ADP-ribosylation).

There is also an irreversible covalent modifications when enzyme

molecule undergoes partial hydrolysis ⇒ a change in enzyme
properties
– Examples:
• Activation of proteases from inactive precursors (zimogene)
- digestion
• Activation of proteases involved in blood clotting,
• Activation of proteases involved in apoptosis (programmed cell
death).
 Nomenclature and Enzyme Classifications

• First enzymes - empiric names, ex. pepsin.

• Then: name - substrate name plus the suffix “ase”. For example:
amylase (acts upon starch – amylos in Greek), lipase (acts upon
lipids), etc.

• In 1961, the International Union of Biochemistry has introduced

the nomenclature and classification of enzymes.
• So, the name of an enzyme is formed of:
substrate name + cofactor name + name of reaction type +
suffix ase.
• For example: L-lactate-NAD+-oxidoreductase.

• Each enzyme is also codified by a number formed of 4 figures and a

prefix: EC - X1X2X3X4
• X1 represents the class of enzyme. There are 6 classes:
– Oxidoreductases (catalyse reactions in which there is transfer of
electrons, hydrogen atoms or oxygen fixing)
– Transferases (catalyse reactions in which there is transfer of atoms or
group of atoms, other than in class 1)
– Hydrolases (catalyse braking of chemical bonds with water help)
– Lyases (catalyse braking of chemical bonds, differently of hydrolysis)
– Isomerases (catalyze reactions that preserve the brute formula of the
compound)
– Ligases (catalyze reaction of formation of new bonds between carbon
and a non-metal, using the energy liberated by ATP).
• X2 represents the enzyme subclass, function of the mechanism of
action.
• X3 – represents enzyme subclass, function of the enzymatic
cofactor.
• X4 – represents the order number in group or subgroup. If it has the
value of 99, that means the enzyme is incompletely characterized.

• For example, L-lactate-NAD+-dehydrogenase

has the code EC = [Link].
I. Oxidoreductases

• catalyze the transfer reaction of electrons or hydrogen, during

reduction-oxidation reactions
• called dehydrogenases or reductases.
• A small part - oxygenases, which fix oxygen on substrate to create
oxygenated functions.
• There are 5 subclasses, according to the:
– oxidized substrate (-CH2OH, -CHO, -CH2NH2, -CH2CH2-)
– enzymatic cofactors (NADP+, FAD or porphyrinic derivatives)
used during the transfer of reducing equivalents.
 Examples:

alcooldehidrogenaza
alcohol dehydrogenase
CH3- CH2 -OH + NAD+ CH3 - CHO + NADH + H+

succinatdehidrogenaza
Succinate dehydrogenase
HOOC CH2 CH2 COOH + FAD+ HOOC CH CH COOH + FADH2

coenzima QH
Coenzyme QH22-cytochrom
-citocrom Cc reductaza
reductase
coenzima QH2 + 2citCoxidat coenzima Q + 2H+ + 2citCredus

peroxidaza
peroxidase
NADPH + H+ + H2O2 NADP+ + 2H2O

catalaza
catalase
H2O2 + H2O2 O2 + 2H2O
 II. Transferases
• catalyze the transfer of radicals or a group of atoms from a donor
molecule to an acceptor molecule.
• subclasses, function of the transferred group:

– Methylases – transfer methyl radicals to different acceptor

subclasses, as: homocysteine, cholamine, noradrenaline, uracil,
etc. methyl radical is found as S-adenosyl-methionine.
noradrenaline + S-adenosyl-Met →
adrenaline + S-adenosyl-homocysteine

– Aminotransferases (transaminases) – transfer an amino group

from an amino acid to an α-ketoacid.

Alanine-aminotransferase
Alanine + α-ketoglutarate pyruvate + glutamate
 – Acyl-transferase – transfer acyl radicals (R – CO)

Glycerol-3-phosphate-acyltransferase
Acyl-CoA + glycerol-3-P acylglycerol-3-P + CoA

– Transfer of glucidic molecules

Glycogen synthase
UDP-glucose + glycogen(n) glycogen (n+1) + UDP

– Phosphotransferases (kinases) – transfer a phosphate

residue –PO3H2 from a donor molecule (ATP, UTP, CTP) to an
acceptor molecule (carbohydrates, lipids, creatine, acids)

hexokinase
Glucose + ATP glucose-6-P + ADP
III. Hydrolases
• catalyze molecules braking and fixation of H and OH from water on
the liberated valences.
• have no coenzymes.
• Function of the substrate nature, there are several subclasses:
– Glycoside hydrolases (glycosidases) – hydrolyze glycosidic
bonds in oligo and polysaccharides (break N- or O-glycosidic
bonds)
Examples: β-glycosidase, maltase, amylase, etc.

– Phosphoric esters hydrolases (phosphatases) – hydrolyze

esters of phosphoric acid.
Example: acid and alkaline phosphatase, glucose-6-
phosphatase, etc.
glucose-6-phosphatase
glucose-6-P + H2O glucose + Pi
– Lipid hydrolases – hydrolyze esters of carboxylic acids.
Triglyceride lipase
Triglyceride + H2O 2 fatty acids +
2-monoacylglycerol

– Peptide and protein hydrolases (peptidases) – hydrolyze

peptide bonds in proteins. Example: peptidases in digestive
juices, in plasma, in conjunctive tissue, cathepsins, etc.

– Nucleotide, nucleoside and nucleic acid hydrolases

(nucleotidases, nucleosidases nucleases) – hydrolyze N-
glycosidic and phosphodiester bonds, forming:
• Nucleic acids → nucleotides
• Nucleotides → nucleosides + ATP
• Nucleosides → ribose + nitrogenous base
IV. Lyases (synthases)
• catalyze reactions in which water, ammonia or carbon dioxide
components are added or eliminated.

– Decarboxylases - eliminate carbon dioxide

– Aldolases – break the bond C3 – C4 in hexoses

– Hydratases and dehydratases – fix or eliminate a water

molecule.

fumarase
Fumaraza
HOOC - CH = CH - COOH + H2O HOOC - CH - CH(OH) - COOH

anhidraza carbonica
Carbonic anhydrase
H2O + CO2 H2CO3
V. Isomerases
• catalyze structural modifications in a compound without the
modification of its molecular formula. Function of the type of
modification, there are:
– Mutases – transfer, intramolecularly, a chemical group:
Phosphoglucomutase
Glucose-6-P glucose-1-P

– Epimerases and racemases – catalyze modifications of

asymmetric carbon atom configuration.
UDP-galactoepimerase
UDP-galactose UDP-glucose

– Functional modifications, for example ketose ↔ aldose

Triosephosphate isomerase
Glyceraldehydes-3-P dihydroxyacetone phosphate
VI. Ligases (synthases)
• catalyze the formation of new bonds C – C, C – N, C – S, C – O, O –
P, using the energy from ATP.

– C – C bond
Pyruvate carboxylase
Pyruvate + CO2 + ATP + H2O oxalacetate + ADP + Pi

– C – N bond
Glutamine synthase
Glutamate + NH3 + ATP glutamine + ADP + Pi
 Utilization of enzymes in medicine
A. Enzymes in Therapeutics
• utilization is limited by the rapid destruction in organism. Destruction may be
delayed by:
– Chemical modification (succinylation of asparaginase increases its existence in
organism from one hour to 17 hours)
– Incorporation into artificial vesicles called liposomes
– Incorporation into RBC
– Connection with polyethylene glycol.
• In recent years enzymes has been used as drugs for treatment of medical
problems. A couple of examples are given below.
• Streptokinase prepared from Streptococcus is useful for clearing the
blood clots. Streptokinase activates plasma plasminogen to plasmin which,
in turn, attacks fibrin to convert into soluble products.
• Asparaginase is used in the treatment of leukemias. It catalyses the
hydrolysis of asparagine to aspartic acid and ammonia. Tumor cells are
dependent on asparagine of the host’s plasma for their multiplication. By
administering asparaginase, the host’s plasma levels of asparagine are
drastically reduced. This leads to depression in the viability of tumor cells.
• Digestive enzymes like trypsin, lipase and amylase, are used to
improve digestion.
B. Enzymes - Diagnosis of Pathology
• cytolytic process releases the cell content into the blood circulation.
• ⇒ blood plasma contains cellular enzymes.
• For example, hepatocytes live a year, so every day 0.3% of liver is destroyed and cell
content is released into plasma.
• Pathological increase of cytolysis may be the effect of:
– Viral or toxic lesions (hepatitis)
– Anoxic lesions due to lack of vascularity (myocardial infarct is associated to
partial necrosis of cardiac muscle)
• In this respect, measuring the activity of marker enzymes can be used in diagnosis.

• Examples:
• a. LDH – lactate dehydrogenase presents 5 isoenzymes that can be separated by
electrophoresis and quantified. LDH1 (α4) and LDH2 are characteristic for myocardial
tissue and thus LDH1 high content in serum can confirm the clinical diagnosis and
EKG of myocardial infarction.
• b. CK – creatine kinase is found as 3 dimeric isoenzymes (CK – muscle, CK –
myocardial, useful in myocardial infarction at 6-8 hours after the event, CK –
cerebral).
• c. Acid phosphatase increase in prostate cancer.
• d. Amylase and trypsin increase in acute pancreatitis.
• e. Transaminases and isocitrate dehydrogenase increase in hepatic diseases.
• f. Alkaline phosphatase increase in obstructive jaundice and bone diseases.
C. Enzymes - Reagents in bio-clinical analysis

• catalytic agents in industrial and medical applications.

• have the advantage of a great specificity, catalyzing the transformation of a
single substrate (or very small number), no side compounds.
• Lots of enzymes are used in clinical laboratories to determine glucose,
triacylglycerols, cholesterol, uric acid, urea, creatine, etc.
• Such enzymes can also be immobilized by binding them to a solid, insoluble
matrix, which will not affect the enzyme stability or its activity. The bound
enzyme can be preserved for long periods without loss of activity.
• Example: glucose oxidase and peroxidase, immobilized and coated on a
strip of paper or plastic, are used in the determination of glucose in plasma
or blood.
Glucoseoxidase
Glucose + O2 Gluconolactone + H2O2

peroxidase
o-tolidine + H2O2 Blue complex + H2O

• The intensity of the blue color depends on the concentration of glucose.

D. ELISA Technique
• type of analysis that uses enzymes bound to immunoabsorbants.
• used for the determination of very small amounts of antigens - 10-8 –
10-10 mol/l (hormones, receptors, viral proteins, etc.).
• The reaction is:
Ag + Ac-E → Ag – AcE

• AC-E – enzyme bound to an antibody

• Ag – antigen to determine
• The complex that is formed, Ag-AcE, is separated by the rest of Ac-
E and is incubated with a chromogenic substrate of the enzyme.
Colour intensity is proportional to the antigen quantity.
Synthetic enzymes

Semisynthetic enzymes –chemical modification of known enzymes

⇒new catalytic properties.
– Example: papain is a proteolytic enzyme; chemical fixation of a flavine
to the cysteine in position 2 suppresses the proteolytic activity and
develops an oxidoreductase activity, previously not found.
– Another method is controlled mutagenesis – a single amino acid is
selectively changed.
Synthetic enzymes – are cyclic structures whose spatial structure is
analogous to an enzymatic site (cyclic polymers of glucose:
cyclodextrins (α, β, γ, after the number of glycosyl residues 6, 7, 8).
Abzymes – are antibodies with catalytic function.
– antibody developed to a stable molecule that's similar to an unstable
intermediate of another (potentially unrelated) reaction,
– the developed antibody will enzymatically bind to and stabilize the
intermediate state, thus catalyzing the reaction.
– Example: Phosphorate is the transition state characteristic to ester bond
in lipids. The obtained antibodies behave like lipase, increasing the
hydrolysis speed 103-105 times. An anomeric stereospecificity type may
also be obtained.