1

Taras Shevchenko National University of Kyiv

The Institute of Biology and Medicine

METHODICAL POINTING
from the course
“Biological and bioorganic chemistry”
(part 7. Enzymes as biological catalysts)

for the studens of a 2 course

with English of educating

Compiler – the candidate of biological sciences,

the associate professor
Synelnyk Tatyana Borysivna

Readers:

It is ratified to printing by meeting of scientific advice of The Institute of Biology

and Medicine (“____”________________ 2018, protocol №____)

Kyiv-2018
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CONTENT
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7. Enzymes as natural biological catalysts

Enzymes are substances (usually proteins but sometimes RNA or

RNA/protein complexes) present in the cell in small amounts that function to
speed up or catalyze chemical reactions (fig. 7.1).
CO2 + H2O → H2CO3
(carbonic anhydrase)

Fig. 7.1. Example of enzymes: carbonic anhydrase as an enzyme that

catalyze chemical transformation CO2 and H2O into H2CO3

A catalyst is defined as "a substance that increases the rate of a chemical

reaction without being itself changed in the process”. In some cases, enzymes
can make a chemical reaction millions of times faster than it would have been
without it.
Now more than 2,000 enzymes are known. These powerful natural
biocatalysts are much more effective than chemical catalysts. They are highly
specific to their substrates, accelerate strictly certain chemical reactions and
function at physiological values of t and pH
Enzymes work by lowering the activation energy required for
reactions to occur. Activation energy is the minimum energy needed to make
a reaction happen. Enzymes lower this amount by providing an alternative
pathway for the reaction that requires less energy to occur. They catalyze
nearly all the chemical reactions taking place in the cells of the body
Some enzymes act as structural components of membranes. In eukaryotic
cells, some enzymes reside in specific organelles; for example, enzymes for
cellular respiration are located in mitochondria, and enzymes for hydrolytic
cleavages are in the lysosomes. Some enzymes functions in blood (coagulaltion
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factors), whereas other are working in liver (arginase), brain (enzymes of
neuromediator metabolism), kidney, skeletal muscles, heart etc.
A number of enzymes acts in a certain sequence and catalyzes hundreds
of multistage reactions (multifunctional complexes). Such complexes work in
reactions of glycolysis, gluconeogenesis, respiratory chain. A number of
enzymes in this sequence are regulatory - they can perceive various metabolic
signals and, accordingly, change oneself activity, resulting in all the enzyme
systems in the cell acting in a coordinated way.

7.1. History of enzymes research

French chemist Anselme Payen was the first to discover an
enzyme, diastase, in 1833. A few decades later, when studying
the fermentation of sugar to alcohol by yeast, Louis Pasteur concluded that
this fermentation was caused by a vital force contained within the yeast cells
called "ferments", which were thought to function only within living organisms.
He wrote that "alcoholic fermentation is an act correlated with the life and
organization of the yeast cells, not with the death or putrefaction of the cells.
In 1877, German physiologist Wilhelm Kühne (1837–1900) first used the
term enzyme, which comes from Greek ἔνζυµον - "in yeast“ (en = in, zyme =
yeast), to describe this process.
Eduard Buchner submitted his first paper on the study of yeast extracts
in 1897. In a series of experiments at the University of Berlin, he found that
sugar was fermented by yeast extracts even when there were no living yeast
cells in the mixture. He named the enzyme that brought about the
fermentation of sucrose "zymase". In 1907, he received the Nobel Prize in
Chemistry for "his discovery of cell-free fermentation".
Following Buchner's example, enzymes are usually named according to the
reaction they carry out: the suffix -ase is combined with the name of
the substrate (e.g., lactase is the enzyme that cleaves lactose) or to the type of
reaction (e.g., DNA polymerase forms DNA polymers).
The biochemical nature of enzymes was still unknown in the early 1900s.
Many scientists observed that enzymatic activity was associated with proteins,
but others (such as Nobel laureate Richard Willstätter) argued that proteins
were merely carriers for the true enzymes and that proteins per se were
incapable of catalysis.
In 1926, James B. Sumner showed that the enzyme urease was a pure
protein and crystallized it; he did likewise for the enzyme catalase in 1937.
The conclusion that pure proteins can be enzymes was definitively
demonstrated by John Howard Northrop and Wendell Meredith Stanley,
who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin.
These three scientists were awarded the 1946 Nobel Prize in Chemistry.
The discovery that enzymes could be crystallized eventually allowed their
structures to be solved by x-ray crystallography. This was first done
for lysozyme, an enzyme found in tears, saliva and egg whites that digests the
coating of some bacteria; the structure was solved by a group led by David
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Chilton Phillips and published in 1965. This high-resolution structure of
lysozyme marked the beginning of the field of structural biology and the effort
to understand how enzymes work at an atomic level of detail.
All enzymes were once thought to be proteins, but since the 1980s the
catalytic ability of certain nucleic acids, called ribozymes (or catalytic RNAs),
has been demonstrated, refuting this axiom.

7.2. Importance of enzyme in organism and industry

Most of biochemical reactions in the living organism are regulated by

enzymes. Enzymes catalyze all aspects of cell metabolism:
• enzymes help the body break down larger complex molecules -
proteins, carbohydrates and fats of food into smaller molecules in
digestive system; glucose is then broken down with the help of several
enzymes. Glycolysis involves around 10 enzymes and the Krebs Cycle involves
another 8. The Electron Transport Chain involves the movement of hydrogen
ions across a membrane to help the production of ATP, using the enzyme ATP
synthase;
• the construction of cellular macromolecules from
smaller precursors.
• DNA replication - each cell in your body contains DNA. Each time
a cell divides, that DNA needs to be copied. Enzymes helicase, DNA
polymerase, ligase help in this process.
• Liver enzymes break down toxins in the body. To do this, it uses
a range of enzymes.
• Without enzymes, many of these reactions would not take
place at a perceptible rate.
• Enzymes are also needed in muscle contraction, conducting a
nerve impulse and blood coagulation
• Enzymes also have valuable industrial applications - for baking
bread, making cheese, alcohol, wine, tea, dyes, organic acids and solvents,
cleaning the waste water, etc.
In this way enzymes have been practiced from earliest times, but not until
the 19th century were these reactions understood to be the result of the
catalytic activity of enzymes.

7.3. Enzymes and diseases

A number of human diseases, in particular, hereditary, are associated

with insufficiency or complete absence of 1 or more enzymes. For example,
such inherited human diseases as albinism and phenylketonuria, result from a
deficiency of a particular enzyme. For the treatment of such conditions, either
the artificial introduction of a specific enzyme (substitution therapy) or the
exclusion of the substrate of this enzyme from the diet (for example, in the case
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of intolerance of lactose - when there is no lactase enzyme - a lactose-free diet)
is used.
Biotechnological methods for "repairing" DNA are developed - then in cells
the ability to synthesize the required enzyme is restored.
There are also diseases caused by excessive activity of certain enzymes -
inhibitors of the corresponding enzymes are used for their treatment.
Enzymes are used for the diagnosis of many diseases - it is important
to measure the activity of a number of enzymes in the plasma (e.g., organ-
specific, indicator enzymes, isoenzymes) and in samples of tissues.
Enzymes can be used therapeutically (for example, digestive enzymes,
enzymes that kill disease-causing microorganisms).

7.4. Chemical nature of enzymes

Enzymes are proteins having catalytic activity, which depend on the

degree of preservation of the native protein structure. When boiling, treating
with alkali, acid, proteolytic enzymes, they are deactivated (enzyme
denaturated), because the tertiary structure of the protein is lost. Even with the
preservation of the secondary and primary structure of the protein, the enzyme
becomes catalytically inactive.
Simple enzymes - consist only of amino acids, that is, they have only the
protein part (pepsin, urease, RNase);
Many enzymes require a nonprotein cofactor to assist them in their
reaction. Such enzymes are called complex enzymes. In this case, the protein
portion of the enzyme, called an apoenzyme, combines with the cofactor to
form the whole enzyme or holoenzyme (fig. 7.2).

Structure of enzymes
Enzymes

Complex or holoenzymes (protein part Simple (only protein)

and nonprotein part – cofactor)

Apoenzyme (protein
part) Cofactor

Prosthetic groups Coenzyme

-large organic
-usually small inorganic molecule
molecule or atom;
-loosely bound to
-usually tightly bound apoenzyme
to apoenzyme
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Holoenzyme= Apoenzyme+ Cofactor

Fig, 7.2. Schematic structures of simple and complex enzymes

The protein part of the enzyme is thermolabile and is responsible for the
specificity of the enzyme; the non-protein part is thermostable and takes part
in binding of the enzyme to the substrate and in the transfer of electrons,
atoms or ions from one substance to another. The complex enzyme without its
non protein part is inactive.

7.4.1. Cofactor, prosthetic group, coenzyme

Cofactor is a direct participant in the catalytic event and thus is required
for enzymatic activity. A cofactor may be:
• coenzyme — an organic molecule, which serve as carriers for
chemical groups or electrons. The next organic compounds can act
as coenzyme:
• Vitamins and their derivatives;
• Nucleotide coenzymes (FAD, FMN, NAD, NADP);
• Hem and other porphyrin coenzymes that contain iron ion
(in hem) or other metal which can change oneself valence
(e.g. Fe2+ → Fe3+) and therefore participate in reactions of
oxidation/reduction (in particular, they are the part of
cytochromes, catalase and peroxidase);
• Peptides (e.g., glutathione - tripeptide glutamynil cysteinil
glycine which may exist in the reduced or oxidized form due
to the presence of SH-groups and which is a coenzyme for
many oxidoreductases):
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• an inorganic metal ions such as Ca2+, Mg2+, and K+;
Some enzymes require both coenzyme and metal ions.
A cofactor may be either tightly (by covalent bond) or loosely (by non-
covalent bond) bound to the enzyme. Cofactor, tightly bound to the
apoenzyme by covalent bonds is called a prosthetic group.

7.4.2. Vitamins and their derivatives as the coenzymes

The next coenzymes are represented by vitamins and their derivatives:

• Thiamine pyrophosphate (TPP) is a derivative of vitamin B1
(thiamine) – it is a coenzyme of pyruvate dehydrogenase
(decarboxylation reaction):

• Flavin mononucleotide (FMN) and flavin adenine dinucleotide

(FAD) are the derivatives of vitamin B2 (riboflavin) - in
oxidation/reduction reactions:

• Nicotinamide adenine dinucleotide (NAD) and nicotinamide

adenine dinucleotide phosphate (NADP) - are the derivatives of
vitamin B5 (nicotinic acid) - in oxidation/reduction reactions:
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• Pyridoxal phosphate, pyridoxamine phosphate - are the

derivatives of vitamin B6 (pyridoxine) - in enzymes of
transamination and decarboxylation of amino acid:

• Coenzyme A (coenzyme of acylation) and pantothein phosphate

(coenzyme of acyl transfer protein of higher fatty acids synthase) - is
a derivative of vitamin B3 (pantothenic acid):
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• Tetrahydrofolic acid (THFA) - is a derivative of vitamin B10 (folic
acid) - it transfers the single-carbon fragments (e.g. -CH2-; -COH;
-CH=):

• Methylcobalamin and deoxyadenosylcobalamin - are the

derivatives of vitamin B12 (cyanocobalamin) that take part in the
synthesis of nucleic acids in the hematopoietic organs (the first - in
the methylation reactions, the second - in the reactions of
intramolecular isomerization):

• Carboxybiotin - is a derivative of vitamin H (biotin) that is used in

the reactions of carboxylation.
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Coenzymes are also represented by such vitamin-like substances as:
• lipoic acid:

• ubiquinone:

• carnitine:
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7.5. Structural and functional particularities of enzymes: active site of

simple enzymes

The active site of enzyme is the area on the enzyme (a special pocket or
cleft) that binds to the substrate with enzyme-substrate (ES) complex forming.
Active site is responsible for:
• specific binding of the enzyme with the substrate,
• formation of the enzyme-substrate complex,
• catalytic transformation of the substrate.
Enzymes are usually very large proteins and the active site is just a small
region of the enzyme molecule. Active site is formed due to the tertiary structure
of the protein (and therefore it is disrupted when denaturation occurs). It
consists of about 15 amino acid residues - mainly such as Ser, Cys, His, Tyr,
Lys, ect., which provide the enzyme both spatial and electrical affinity for the
substrate.
Because of these amino acids presence, the enzyme active site has a lot of
such functional groups as:
• ‒OH-groups of serine, threonine, tyrosine;
• imidazole rings of histidine;
• ‒SH-group of cysteine;
• ‒COOH-group of glutamine and asparagine;
• ‒NH2-groups of arginine and lysine (Fig. 7.3).

Charged
Nonpolar
Polar

OH
HO
HO
+
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Fig. 7.3. Three main types of functional groups in enzyme active site

These functional groups take part in enzyme-substrate complex formation

and in catalysis process.
Active site has very specific 3-D Shape with a specific arrangement of
functional groups. It is flexible.
There are two parts in the structure of the active site (Fig. 7.4):
- catalytic site (it takes part in substrate transformation);
- binding site - which binds the enzyme to the substrate.

Binding site
Active site
Catalytic site

Fig. 7.4. Catalytic and binding sites as the parts of enzyme active site

The active site of complex enzymes also contains a cofactor and a part of
the apoenzyme that spatially adjoins it. The cofactor is responsible for the
formation of the enzyme-substrate complex, the formation of the tertiary or
quaternary structure of the apoenzyme, the catalytic transformation of the
substrate
An enzyme may have 1, 2, 3 or more active sites, which depends on the
number of subunits in the enzyme structure.

7.6. Isoenzymes

Isoenzymes are enzymes that catalyze the same reaction, but are
contained in different tissues and differ in a number of physico-chemical
properties (electrophoretic mobility, stability). The basis of these differences is
the genetically determined difference in primary structures of their protein
molecules. In addition, some of the isoenzymes differ in subunit composition
(e.g., lactate dehydrogenase, creatine kinase, alkaline phosphatase).
Some of enzymes that exist in different isoenzyme forms are the next:
Lactate dehydrogenase (LDH) - makes the conversion of lactic acid to
pyruvic acid; it has 5 isoenzymes - each of them consists of 4 subunits of H- or
M-type:
• in the heart, kidneys, brain, red blood cells - LDH1 (H4) and
LDH2 (MH3);
• in the liver and skeletal muscles - LDH4 (M3H) and LDH5 (M4);
• in the lungs, spleen, lymph nodes - LDH3 (M2H2).
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7.7. The properties of enzymes as catalysts

7.7.1. The common properties to all types of catalysts

They are the next:
• these biological catalysts accelerate only those reactions that are
possible in terms of thermodynamics (moving toward thermodynamic
equilibrium);

• they don’t change the direction of the reaction;

• they increase the speed of approaching the system to the

thermodynamic equilibrium, without changing the equilibrium
point;

• they don’t relatively change after the reaction;

• they operate in relatively small quantities.

7.7.2. Pecularities of enzymes as catalysts

7.7.2.1. The rate of catalytic activity and conditions of enzymatic

reactions. Enzyme activity.
Enzymes have a significantly higher catalytic activity. For example,
reaction:
H2O2 → H2O +O2

• uncatalyzed – months
• with Fe ions - 30,000 x faster
• with catalase - 100,000,000 x faster
Mild reaction conditions - t° = 37℃, physiological pH, ambient
atmospheric pressure
Enzymes are never expressed in terms of their concentration (as mg or µg
etc.), but are expressed only as their activities - the rate of appearance of
product or the rate of disappearance of substrate

7.7.2.2. Specificity of enzymes. Enzymes have specificity of action (on

a specific substrate, on a group of similar structure substrates certain type of
bond in the molecule) due to conformational and electrostatic complementarity
of enzyme and substrate and structural features of the active center.
There are the next types of enzyme specificity:
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• bond specificity (also called relative specificity – enzyme acts on
identical bonds in the substrate. It is typical mainly for digestive
enzymes, for example, proteinases, lipases, phosphatases:
− amylase can hydrolyze α-(1→4) glycosidic linkage in starch and
glycogen (it functions in mouth as well as in intestine);
− lipase can hydrolyze ester bond between glycerol and fatty acids
in any fats (it works in intestine);
− proteinases hydrolize all peptide bonds formed by any amino
acids (they work in stomach and intestine).
• group specificity (also called moderate, or structural specifity) -
enzyme is specific to type of bonds and group of atoms surrounded it.
The classical examples of group specificity are represented by
endopeptidases and exopeptidases of digestive tract – e.g.:
− pepsin hydrolyzes a peptide bond in which amino group is
contributed by aromatic amino acids – for example, phenyl alanine,
tyrosine or tryptophan, it works in stomach:

− trypsin hydrolyzes a peptide bond in which amino group is

contributed by basic amino acids – lysine, arginine or hystidine –
and works in intestine:

− chymotrypsin hydrolyzes a peptide bond in which amino group

is contributed by aromatic amino acids – for example, phenyl
alanine, tyrosine or tryptophan (acts like pepsin, works in
intestine):
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− aminopeptidases hydrolyze a peptide bond from N-terminus of

protein, work in intestine
:

− carboxypeptidases hydrolyze a peptide bond from C-terminus of

protein, work in intestine too:

• absolute (substrate) specificity – enzyme is specific to only one

substrate and one reaction. For example:
− urease catalyzes the conversion of urea, without affecting the
methyl urea;
− lactase, maltase and sucrase catalyze hydrolysis of lactose,
maltose and sucrose, respectively
• stereospecificity (optical) specificity – enzyme acts on one of
stereoisomers (eg., L- or D- isomer). For example:
− enzymes which convert carbohydrates act on D-isomers;
− fumarase affects trans-isomer (fumaric acid) without affecting
the cis-isomer (maleic acid);
− D-amino acid oxidase acts on only D-amino acids;
− L-amino acid oxidase acts on only L-amino acids:
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− α-glycosidic bonds of starch and glycogen are hydrolyzed only
by α-glycosidase (α-amylase);
− β-glycosidic bonds of cellulose are hydrolyzed only by β-
glycosidase (β-amylase):

7.7.2.3. Dependence of the rate of enzymatic reaction on temperature.

For chemical reactions with a nonbiological catalyst, with a 10°C rise
in temperature the reaction rate doubles (Q10 = 2, the temperature coefficient
of Van Hoff);
Enzyme-controlled reactions follow this rule as they are chemical
reactions. BUT at high temperatures proteins denature. Therefore for
fermentative reactions this is true only in the range of low temperatures
(around 40 degrees Celsius). Above these temperatures the enzymes will start to
denature (unfold) - enzyme changes shape; this alters the shape of the active
site and its activity decreases or disappears.
Just before the point of denaturation is the 'optimum temperature' where
the rate of reaction will be the highest. The optimum temperature for an
enzyme controlled reaction will be a balance between the Q10 and
denaturation.

Optimum
Increasing
number of Denaturation
collisions (Q10)
Enzyme
activity

0 10 20 30 40 50
Temperature / °C
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Fig. 7.5. Dependence of the rate of enzymatic reaction on temperature

In cold temperatures, enzymes work slower due to the lack of energy they
can get (lack of heat). Therefore, the low temperatures inhibit the enzyme
without destroying its structure.
The optimum temperature for the most enzymes in the human body is
around 37°C - body temperature (Fig. 7.6).
The rate of enzymatic
reaction

Fig. 7.6. The optimum t° of the most human enzymes

The examples of thermostable enzymes - alkaline phosphatase of the liver

(active at 50 ° C), myokinase (100 ° C). A few bacteria have enzymes that can
withstand very high temperatures up to 100°C. In contrast, cold water fishes
will die at 30°C because their enzymes denature

7.7.2.4. Dependence of the rate of enzymatic reaction on the pH of

medium
Depending on the pH, the enzyme may be in an ionized or nonionized form,
which is reflected on the tertiary structure of the protein, and, consequently,
on the formation of the active site of the enzyme. The pH also affects the
ionization state of the cofactor (or prosthetic group) and the substrate.
When the enzyme is in a pH that is classed as 'extreme' either side of its
optimum pH, the enzyme will denature. For most enzymes optimum pH 6-8
(Fig. 7.7).
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Fig. 7.7. The dependence of the rate of enzymatic reaction on the pH of medium

Glucose 6-phosphatase of hepatocytes (liver cells), with a pH optimum of

about 7.8, is responsible for releasing glucose into the blood. The normal pH of
the cytosol of hepatocytes is about 7.2.
Enzymes in the intestines work best at alkaline pH (trypsin - 8-9),
whereas enzymes in the stomach work best at acidic pH (pepsin - 1,5-2,5)
(Fig. 7.8).

Fig. 7.8. The dependence of the rate of some enzymatic reaction on the pH of medium
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7.7.2.5. Dependence of the rate of enzymatic reaction on the enzyme
concentration.
Other factors affecting the rate of enzymatic reaction is the enzyme
concentration. The more enzymes present and exposed to available substrates,
the faster the overall reaction rate (Fig. 7.9).

Fig. 7.9. The dependence of the rate of enzymatic reaction on the enzyme concentration

So the initial rate of an enzyme-catalyzed reaction is always

proportionate to the concentration of enzyme. This property of enzyme is
made use in determining the serum enzyme for the diagnosis of diseases.

7.7.2.5. Dependence of the rate of enzymatic reaction on substrate

concentration. The rate of reaction increases as substrate concentration
increases (at constant enzyme concentration) (Fig. 7.10).

Fig. 7.10. The dependence of the rate of enzymatic reaction on the substrate concentration
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Maximum activity occurs when the enzyme is saturated (when all enzymes
are binding substrate).
The comparison of the dependence of the reaction rate on the substrate
concentration for enzymatic and non-enzymatic reactions is represented on Fig.
7.11.

Non-enzymatic reactions Enzymatic reactions

Faster reaction but it reaches a

saturation point when all the
The increase in velocity is proportional enzyme molecules are occupied
to the substrate concentration.
•
Fig. 7.11. The comparison of the dependence of the reaction rate on the substrate
concentration for enzymatic and non-enzymatic reactions

7.7.2.6. Cofactor availability

In some cases, enzymes won't work without a co-factor - these can be
vitamins or minerals, so a lack of particular vitamins or minerals can cause
enzymes to not work to their potential. For instance, carbonic anhydrase, an
enzyme that helps maintain the pH of the body, cannot function unless it is
attached to a zinc ion:
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7.8. Enzymes nomenclature

The most enzymes have systematic and trivial (working) names.

To generate the trivial (working) name of an enzyme, the suffix -ase is
added to:
- the name of its substrate:

substrate name: + ase

For example, lactase is the enzyme that cleaves lactose; sucrase catalyzes
the hydrolysis of sucrose; urease, which catalyzes the breakdown of urea) -
because of these enzymes have an absolute specificity

- both the name of substrate and the type of reaction:

substrate name + name of the chemical reaction type + ase

Therefore, enzyme that catalyzes reaction of dehydrogenation of lactate

(hydrogen deleting from lactate, or its oxidation) is called lactate
dehydrogenase (from lactate + dehydrogenation).
Systematic name of enzyme is generated by the principle:

substrate name : name of the chemical reaction type + ase

So, the systematic name of lactate dehydrogenase will be:

Lactate : NAD oxidoreductase

(lactate is substrate I, NAD is substrate II, oxidoreductase - an enzyme involved

in an oxidation-reduction reaction).
Sometimes historical (common) names are used, particularly for the
digestion enzymes such as pepsin and trypsin.

7.9. Classification of enzymes

Classification of enzymes is based on the type of chemical reaction which

they catalyze. All enzymes are divided on 6 classes:
1. oxidoreductases (they are involved in oxidation-reduction reactions);
2. transferases (transfer a chemical group from one substance to
another);
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3. hydrolases (cleave the substrate by uptake of a water molecule
(hydrolysis));

4. lyases catalyze breaking down (an "elimination" reaction) of various

chemical bonds (C-C, C-N, C-O, C-S bonds) by means other than
hydrolysis and oxidation, often forming a new double bond or a new
ring structure;
5. isomerases (isomerization reaction - transfer a group within a
molecule to form an isomer);
6. ligases (or synthetases) (they form C-C, C-N, C-O, C-S bonds and couple
the formation of these bonds to the breakdown of a pyrophosphate bond
in adenosine triphosphate (ATP) or a similar nucleotide triphosphates.
These classes are divided on subclasses. Subclasses clarify the action of
the enzyme via indicating the nature of the substrate; there are also sub-
subclasses.

7.9.1. Oxidoreductases

These enzymes catalyze the reactions of oxidation and reduction (oxidation

– deletion of electrons (protons) from the substrate; reduction - adding of
electrons (protons) to the substrate).
The reaction of lactate oxidation, catalyzed by lactate dehydrogenase, is
the example of such reactions:

Alcohols, ketones, acids, aldehydes, compounds with NH2-, NH-, SH-

groups are uzed as substrates-donors of electrons or protons, whereas
coenzymes NAD+, NADP, FMN, FAD, metal ions and disulphide compounds
play role of substrates-acceptors of electrons or protons.
Systematic names of this class enzymes are formed by the scheme:

donor : acceptor-oxidoreductase.

By trivial name enzymes of this class are divided into:

- dehydrogenases (cleave protons or electrons from the oxidation
substrate and transfer them to any acceptor except oxygen and hydrogen
peroxide);
 24
- oxidases (acceptor - oxygen);
- peroxidases (acceptor - hydrogen peroxide);
- reductases (have a more reducing effect);
- oxygenases (or hydroxylases) - facilitate the direct inclusion of
O2 into substrate.
Examples of this class enzymes are cytochrome oxidase, catalase,
peroxidase, lactate dehydrogenase, glucose oxidase, phenylalanine
hydroxylase.

7.9.2. Transferases

These enzymes transfer a chemical group from one substance to another.

The reaction of transamination, catalyzed by alanine aminotransferase, is the
example of such reactions:

The enzymes of this class are divided on 8 subclasses depending on the

type of transferring groups, including:
- methyltransferases (-CH3);
- aminotransferases (-NH2);
- acyltransferases (fatty acids residues);
- carboxyransferases (-CO-);
- glycosyltransferases (carbohydrate residues)
- sulfotransferases (sulfuric acid residue)
- phosphotransferases (or kinases) - phosphoric acid residues
on ADP or ATP molecule on the substrate.

Systematic names of this class enzymes are generated by the scheme:

donor : acceptor-transport group-transferase.

For example, hexokinase (trivial name) is ATP : D-hexose-6-

phosphotransferase (enzyme that carries the phosphoric acid residue
 25
(phosphorylation reaction) from ATP to any hexose to the position near the
sixth carbon atom of this hexose).

Representatives of enzymes of this class are:

• enzymes involved in the disinfection of natural and foreign toxins
(glucuronyl transferase);
• enzymes used in the diagnosis of acute hepatitis and myocardial
infarction (alanine aminotransferase (ALT) and aspartate
aminotransferase (AST), involved in transamination reaction);
• creatine kinase – is involved in creatine phosphorylation reaction
and is used in the diagnosis of lesions of skeletal muscle;
• protein kinases (are involved in protein phosphorylation reaction)

7.9.3. Hydrolases

Hydrolases (esterases, peptidases, glycosidases, ect.) catalyze the

hydrolysis of various bonds with adding water molecule across a bond:

R - X + HOH → ROH + HX

Examples of enzymes of this class are:

• enzymes of the gastrointestinal tract (lipases, proteases, amylase,
maltase, lactase);
• enzymes of lysosomes;
• deaminases;
• phosphatases.

7.9.4. Lyases

They catalyze the breakdown of C‒C, C‒N, C‒O, C‒S bonds; in this regard,
they are divided into subclasses:

• С‒С bond – decarboxylases of amino and keto acids;

• С‒О bond – dehydratases, or hydratases (for example, carbonic

anhydrase, which breaks down H2CO3 with СО2 and Н2О forming);

• С‒N bond – for example, argininosuccinate lyase - during the formation

of urea it decomposes argininosuccinate into fumaric acid and arginine;

Aldolases which catalyze the breakdown of hexosophosphates with two

trioses forming also belong to lyases:
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7.9.5. Ligases (synthetases)

These enzymes form C-C, C-N, C-O, C-S bonds. There are 5 subclasses of
ligases.

Examples of this class enzymes are:

• aminoacyl-tRNA synthetase;
• acetyl CoA synthetase;
• acetyl~CoA carboxylase;
• glutamine synthetase:

7.9.6. Isomerases

These enzymes catalyze isomerization reaction - transfer a group within a

molecule to form an isomer. They are divided into subclasses:
 27
• racemases (for example, convert L-amino acids into D-amino
acids):

• epimerases (are involved in mutual conversion of sugars, for

example, galactose in glucose or glucose-6-phosphate into fructose-
6-phosphate;

• mutases (take part in transfer of chemical groups from one part of

the molecule to another; they often need for coenzyme – vitamin
B12 derivative; example – phosphoglucomutase;

• cis-trans-isomerases.

7.10. Principle of the international classification

Each enzyme has classification number consisting of four digits. For

example:

EC: 1.1.1.1 - ALCOHOL DEHYDROHENASE

EC: 2.7.1.1 - HEXOKINASE

In this notation:
• 1 digit means class;
• 2nd digit – subclass;
• 3rd digit (sub-subclass);
• 4th digit (serial number of the enzyme in the sub-subclass).

Therefore, EC: 1.1.1.1 - the systematic name of this enzyme is alcohol :

NAD+-oxidoreductase; its trivial name is alcohol dehydrogenase. This
notation means that this enzyme belongs to:
• Class: 1 - oxidoreductase
• Subclass: 1.1 - act on the CH-OH group of substrate-donor
• Sub-subclass 1.1.1 - use NAD+ or NADP+ as acceptors
• The serial number of this enzyme in the sub-subclass is 1
 28
This enzyme catalyzes the next type of reaction:

Alcohol + NAD+ → Aldehyde + H+ + NADH

For example:

CH3CH2OH + NAD+ → CH3CHO + NADH + H+

For enzyme with EC: 2.7.1.1 - its systematic name is ATP : D-hexose-6-
phosphotransferase and its trivial name is hexokinase. This notation means
that this enzyme belongs to:
• Class: 2 - transferase
• Subclass: 2.7 – transfer of phosphate
• Sub-subclass 2.7.1 – alcohol is phosphate acceptor
• The serial number of this enzyme in the sub-subclass is 1

This enzyme catalyzes the next reaction:

Glucose + ATP glucose-6-P + ADP

6 CH 2OH 6 CH OPO 2−
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H 2+ OH
Mg
OH OH OH OH
3 2 3 2
H OH Hexokinase H OH
glucose glucose-6-phosphate

Therefore enzymes are classified into six functional classes (EC number
Classification) by the International Union of Biochemists (I.U.B.) on the basis of
the types of reactions that they catalyze:
• EC 1. Oxidoreductases
• EC 2. Transferases
• EC 3. Hydrolases
• EC 4. Lyases
• EC 5. Isomerases
• EC 6. Ligases

7.11. Some important groups of enzymes

Constitutive enzymes - they are constantly present in certain organism

and constantly act to catalyze corresponding reactions;
 29
Adaptive (or inducible) enzymes - they are not normally present or there
are very few of them and they appear or their quantity increases due to the
adaptation of the organism to certain conditions. Substances causing the
formation of such enzymes are called inductors. For example, in animals,
sucrose induces the formation of sucrase that breaks down this sugar to 2
monomers; macrophagal NO-synthase (isoform iNOS) is induced by
compounds of bacterial origin (for example, lipopolysacharide) - it is necessary
for the destruction of bacteria. Excessive NO formation by iNOS under bacterial
infection condition is one of the reasons of septic shock development. NO,
generated by iNOS, leads to blood pressure drop and damage of cells and
tissues.
Organ-specific enzymes (eg, arginase, glucokinase in liver) – are used in
diagnosis.
Indicative enzymes - they are not organ-specific, but in combination with
other enzymes are also used in the clinic, because their intracellular
concentration is greater than their content in the blood plasma, and under
cytolysis condition (in cell destruction), their plasma content significantly
increases (eg, ALT , AST, LDH).

7.12. How enzymes work

7.12.1. Activation energy

In any substance, individual molecules vary greatly differ in the amount of

energy they contain (high, low, most of the molecules - average).
Every chemical reaction between molecules involves bond breaking and
bond forming. The initial energy needed to start a chemical reaction is called
the free energy of activation, or activation energy (ЕA):

A B

C D
Transition state

A B EA
Free energy

C D
Reactants
A B
∆G < O
C D

Products
Progress of the reaction
 30
Activation energy is the amount of energy in calories needed to ensure that
all molecules of 1 mole of a substance at a certain temperature reach
transition state.
According to another definition, the activation energy is the additional
energy required for the transition of the substrate molecules to a transition
(activated) state that precedes their transformation into reaction products.
The rate of any reaction is directly proportional to the concentration of
molecules in the transition state that corresponds to the activation of the
molecule.
In non biological systems activation energy is often supplied in the form
of thermal energy that the reactant molecules absorb from their
surroundings:

Temperature 1 Temperature 2
T2 > T1 Molecules with
sufficient
Energy (~40%)

Molecules with
sufficient
Energy (<5%)
ЕA

In biological systems it is no possible to increase temperature – so

ENZYMES make reactions easier to occur at reasonable temperature by
LOWERING the ACTIVATION ENERGY ЕA of the reaction

So enzymes speed up the rate of chemical reactions because they

lower the energy of activation (ЕA) of biochemistry reaction

For example, for reaction catalyzed by the enzyme catalase:

2H2O2 → O2 + 2H2O

EA = 18 kcal / mole;
in the presence of a chemical catalyst (platinum) EA = 12 kcal / mole;
in the presence of the catalase enzyme EA = 5 kcal / mole.
 31
Enzymes lower the energy of activation by forming an enzyme-
substrate complex allowing products of the enzyme reaction to be formed
and released:

An enzyme-
substrate
complex

7.12.2. Enzyme-substrate complex

The enzyme at the intermediate stage of the reaction interacts with the
reaction substrate to form an enzyme-substrate complex whose transition
state corresponds to much lower EA compared with the transition state of the
reagent.
 32

Substrate

Active site

Enzyme Enzyme-substrate
complex
(a) (b)

On the later stages of reaction this complex is broken with product of

reaction and free enzyme releasing
Binding of substrate to the active site of the enzyme with enzyme-
substrate complex formation can lower an EA barrier due to the interaction of
the substrate with the active site of the enzyme, which creates the spatial
conditions necessary for the implementation of specific acts of catalysis and is
accompanied by:
– orienting substrates correctly;
– straining substrate bonds;
– providing a favorable microenvironment;
– covalently bonding to the substrate.
The enzyme-substrate complex is stabilized by:
• disulfide bonds;
• ionic bonds;
• hydrogen bonds;
• hydrophobic interactions.

7.12.3. Enzyme-substrate complex formation hypothesis

The hypothesis of Warburg and Bailey, or an adsorption hypothesis:

the surface of the enzyme is a place for the absorption of reagents; the number
of molecules per unit of enzyme area increases, and consequently, according to
the law of the active masses, the rate of reaction increases. But this
hypothesis does not explain the specificity of enzymes.
The "lock and key" model, or hypothesis of conformity was firstly
proposed in 1894 by E. Fischer:
 33

In this model:

the active site has a rigid shape (conformation)

rigid spatial correspondence of the substrate and the active site of the
enzyme is the basis of enzyme specificity; the reaction is possible
only when the spatial substrate approaches the enzyme as the
key to the lock

the substrate is a key that fits the lock of the active site

This is an older model, that explains the loss of activity when enzymes
denature. The disadvantage is that it can not explain different types of
specificity, i.e. it is difficult to imagine a situation in which several keys
(substrates) are suitable for the one lock (enzyme)
 34
Induced Fit Model, or Koschland hypothesis - the hypothesis of induced
adjunction of the enzyme to the substrate. In this hypothesis:

• the configuration of the enzyme and its active site is flexible (not
rigid!) and elastic and changes under the influence of the substrate;

• therefore the substrate induces changes in the configuration of the

enzyme molecule in accordance with its own structure (this if "lock
slit“ was made of elastic material and acquired a form corresponding to
the key):

• This model proposes that the initial interaction between enzyme and
substrate is relatively weak, but that these weak interactions rapidly
induce conformational changes in the enzyme that strengthen binding.

• This model is more consistent with a wider range of enzymes and

explains a greater range of substrate specificity

Comparison of Lock and key and Induced fit models is gien on Fig. 7.12.

Fischer: Lock
and key

Koshland:
Induced fit

Change of enzyme and substrate conformations due to

enzyme/substrate complex formation

Fig. 7.12. Comparison of Lock and key and Induced fit models
 35
7.13. Enzymatic reaction steps

In 1913 Michaelis and Menten: when a substrate (S) fits properly in an

active site, an intermediate short-lived compound called enzyme-substrate
(ES) complex is formed:

E + S ES

Within the active site of the ES complex, the reaction occurs to convert
substrate to product (P):

ES → E + P

Enzymes ORIENT Substrates

always in productive orientation Enzymes cause BOND STRAIN
(physical strain and chemical strain
- destabilize existing bonds
“nutcracker effect”
HO
OH OH
OH HO
HO
- HO
+
OH OH
OH HO
HO -
+

The products are then released, allowing another substrate molecule to

bind the enzyme.

This cycle can be repeated millions (or even more) times per minute.
The overall reaction for the conversion of substrate to product can be written
as follows:
E + S ES → E + P

The overall catalytic cycle of an enzyme is represented on Fig. 7.13.

 36
1 Substrates enter active site; enzyme
changes shape so its active site 2 Substrates held in
embraces the substrates (induced fit). active site by weak
interactions, such as
hydrogen bonds and
ionic bonds.

3 Active site (and R groups of

Substrates its amino acids) can lower EA
Enzyme-substrate
complex and speed up a reaction by
• acting as a template for
substrate orientation,
• stressing the substrates
6 Active site and stabilizing the
Is available for transition state,
two new substrate • providing a favorable
Mole. microenvironment,
• participating directly in the
catalytic reaction.
Enzyme

5 Products are
released. 4 Substrates are
converted into
products.
Products

Fig. 7.13. The catalytic cycle of an enzyme

7.14. Activators and inhibitors of enzymes

7.14.1. Enzyme activators

Enzyme activators are molecules that bind to enzymes and increase their
activity. They can be represented by:
• Inorganic ions
- metal ions，such as Na+, K+, Mg2+, Ca2+, Cu2+, Zn2+, Fe2+ et al
- anions, such as Cl‒, Br‒, I‒、CN‒ et al
• Organic compounds
- reducing agents, such as Cys、GSH
- some proteins

7.14.2. Enzymes inhibitors

Inhibitor is any molecule which acts directly on an enzyme and therefore

lower catalytic activity of enzyme. Some enzyme inhibitors are normal body
metabolites. Other may be foreign substances, such as drugs or toxins. The
 37
action of inhibitors is specific to certain enzymes or enzyme groups; inhibitors
act in low concentrations
Concentrated acids, alkalis, salts of heavy metals in high concentrations
also reduce the activity of enzymes - but non-specific due to their denaturation,
acting as denaturants. So inhibitor does not cause denaturation of enzyme.
Inhibition can be:
• reversible: E + I ↔ EI (may be competitive and noncompetitive);
• irreversible: E + I → EI
(E – enzyme, I – inhibitor).

7.14.2.1. Reversible competitive inhibition

Competitive inhibitors are structurally similar to the substrate and
interact with the same active center on the enzyme as the substrate.

E + S ↔ ES → E + P

E + I ↔ EI →
(E – enzyme, I – inhibitor, S – substrate).
Examples:
1. Succinate (HOOC-CH2-CH2-COOH) → fumarate (enzyme succinate
dehydrogenase); malonate (HOOC-CH2-COOH) is structurally very
similar to succinate and forms with enzyme complex that is not capable
to oxidize.
2. Other examples of competitive inhibitors: antivitamins, substances
that are similar in structure to amino acids, purine and pyrimidine
bases and nucleotides. Some antimetabolites, due to their inhibitory
effect on enzymes, are used as antibacterial agents (sulfanilamides,
antibiotics), cytostatics.
How to overcome inverse competitive inhibition? The content of the
substrate must be increased in an incubation environment.

7.14.2.2. Reversible non-competitive inhibition

Such inhibitors are not structurally similar to the substrate, don’t
react with active site of the enzyme molecule, but react with other its
part. They can interact with both the free enzyme (E + I ↔ EI) and the enzyme-
substrate complex (ES + I ↔ EIS). Such inhibition is done by intermediate
metabolism products in living organisms. The addition of a non-competitive
inhibitor to the enzyme reduces its activity, but does not affect the affinity of
the enzyme to the substrate. Such inhibition can not be abolished by
increasing the substrate concentration.
The comparison of competitive and non competitive inhibition is given on
Fig. 7.14.
 38
(a) Normal binding (b) Competitive inhibition (c) Noncompetitive
inhibition
Substrate

Active
site
Competitive
inhibitor

Enzyme

Noncompetitive
inhibitor

Fig. 7.14. The comparison of competitive and non competitive inhibition

7.14.2.3. Irreversible inhibition

It occurs due to destruction or irreversible chemical modification of

the one or more functional groups of the enzyme. Such irreversible
inhibitors are cellular poisons – for example, cyanides, organophosphorus and
thiol poisons. Thus, the alkylating agent iodoacetamide reacts irreversibly with
catalytically active SH-groups:

E-SH + ICH2-CO-NH2 → E-S-CH2-CO-NH2 + HI

The inhibitory effect of the organophosphorus compound diisopropyl

fluorophosphate (DFP) on on the acetylcholinesterase, trypsin, chymotrypsin,
elastase, phosphoglucomutase activities is due to the fact that this compound
irreversibly reacts with catalytically active OH group of serine residue in the
active site:
 39
7.14.2.4. Some specific types of inhibition
"Inhibition by reaction products" - since the reaction product is often
structurally similar to the substrate and, when accumulated, acts as a
competitive inhibitor of the enzyme.
"Inhibition of excess of substrate" - the reason is that the molecules of
the substrate due to mutual interference occupy the wrong spatial position in
the active site of the enzyme, which prevents the normal course of the reaction.
"Genetic inhibition" - the reason is inhibition of the formation of the
enzyme at the transcriptional level, when there is no need for this enzyme.
In feedback inhibition, the end product of a metabolic pathway shuts
down the pathway. Feedback inhibition prevents a cell from wasting chemical
resources by synthesizing more product than is needed.

7.15. The general principles of regulation of activity of enzymes

There are next main types of regulation of activity of enzymes:

1. Alosteric regulation of the enzyme activity (for allosteric enzymes).
Allosteric enzymes are regulated by molecules called effectors
(modifiers) that bind non-covalently at a site other than the active site. Such
factors are products of enzymatic reactions, some substrates, hormones,
nervous system mediators, metals.
Alosteric site is a place of regulatory factors (effectors) influence on the
enzyme. Their attachment to the alosteric site changes the enzyme active
center conformation, which increases or decreases the activity of the enzyme.
The enzyme may also have several alosteric sites, depending on the
number of subunits.
The alosteric and active sites of the enzyme are spatially separated.

2. Regulation at the transcription level - induction or repression of

enzyme biosysnthesis by affecting the genome (via transcriptional factors or
steroid hormones actions);

3. Regulation at the translation level – changes in ribosomal protein

synthesis;

4. Posttranslation regulation:
a) regulation of the activity of the enzyme by its covalent
modification:
− phosphorylation/dephosphorylation (by serine/threonine or
tyrosine protein kinases and protein phosphatases) - addition
or removing of phosphate group:
 40

The regulatory modification of enzymes by

Phosphorylation
Some enzymes are active in
phosphorylated state and inactive in
dephosphorylated one
Active
Inactive

Non- Phosphorylated
phosphorylated Inactive
protein Active
protein
Other enzymes are active in
dephosphorylated state and
phosphorylation will inactivate them

− methylation – the addition of –CH3 group;

− adenylation – the addition of adenylate residue;
− acylation – the addition of fatty acid residue;
− izoprenylation – the addition of one or more isoprene group;
− nitrosylation – the addition of NO group;
− ADP-ribosylation – the addition of ADP-ribose residue;
− proteolytic cleavage - some enzyme are synthesized as inactive
precursor, called zymogens, that are activated by proteolysis
(e.g., digestive enzyme, pepsinogen is inactive and cleaved to
pepsin which is active )
− oxidation of functional groups in the active center;

b) dissociation of the inactive oligomeric enzyme on subunits, of

which one or more exhibits the activity of the enzyme;
c) Location within the cell: Many enzymes are localized in specific
organelles within the cell. This, compartmentation helps in the
regulation of the metabolic pathway.
d) activation or inhibition of enzyme activity using special
regulatory proteins (calmodulin in combination with Ca2+, proteinase
inhibitors);
 41
7.16. Units of activity of enzymes

The rate of enzymatic reaction can be expressed either by the rate of

substrate elimination, or by the rate of reaction product formation.
One international unit (IU, EU or U) is the amount of enzyme consuming
or forming 1 µmol sub-strate or 1 µmol product per minute (µmol / min) under
standard conditions.
In the SI system, the unit of enzymatic activity is 1 katal, corresponding to
the amount of enzyme converting 1 mol substrate per second (1 mol / s).
Relationship between these units:

Standart conditions for determining the activity of enzymes:

− temperature – 25 - 37 ° С;
− pH (according to the enzyme that is determined);
− complete saturation of the enzyme by the substrate.
In practice, derivative units are also used:
The specific activity of an enzyme is the activity of an enzyme per
milligram of total protein (U / 1 mg, expressed in µmol / (min x mg). In the SI
system – katal / 1 kg. Specific activity gives a measurement of enzyme purity
in the mixture.
Turnover number (= molar activity) - the number of moles of substrate
converted to product per mole of enzyme per second. For example, carbonic
anhydrase has a turnover number of 400,000 to 600,000 s−1, which means
that each carbonic anhydrase molecule can produce up to 600,000 molecules
of product (bicarbonate ions) per second

7.17. Enzymes in clinical diagnosis

An enzyme test is a blood test or urine test that measures levels of

certain enzymes to assess how well the body’s systems are functioning and
whether there has been any tissue damage.
Common enzymes used for clinical diagnosis include:
− alanine and aspartate aminotransferases (ALT and
AST);
− alkaline phosphatase;
− amylase;
− creatine kinase;
− lactate dehydrogenase.
 42
Enzyme activity in enzyme tests is given as amount of moles of substrate
converted to product per unit time (more often) or amount of moles of product
formed from substrate per unit time.

7.18. Enzyme assays

Enzyme assays are laboratory procedures that measure the rate of

enzyme reactions. Since enzymes are not consumed by the reactions they
catalyze, enzyme assays usually follow changes in the concentration of either
substrates or products to measure the rate of reaction.
There are many methods of measurement:
− spectrophotometric assays observe change in
the absorbance of light between products and reactants;
they allow the rate of the reaction to be measured
continuously;
− radiometric assays involve the incorporation or release
of radioactivity to measure the amount of product made
over time. An analogous approach is to use mass
spectrometry to monitor the incorporation or release
of stable isotopes as substrate is converted into product.\
− the most sensitive enzyme assays use lasers focused
through a microscope to observe changes in single
enzyme molecules as they catalyse their reactions. These
measurements either use changes in
the fluorescence of cofactors during an enzyme's reaction
mechanism, or of fluorescent dyes added onto specific
sites of the protein to report movements that occur
during catalysis
These studies are providing a new view of the kinetics and dynamics of
single enzymes, as opposed to traditional enzyme kinetics, which observes the
average behaviour of populations of millions of enzyme molecules.