BIOCHEMISTRY

ENZYMES
 Enzymes are macromolecular biological
catalysts which are responsible for metabolic
processes that sustain life.
Enzymes are highly selective catalysts,
greatly
accelerating both the rate and specificity
of metabolic chemical reactions, from the
digestion of food to the synthesis of DNA.
 Enzyme (human glucolase)
Zinc ions that are needed for the enzyme to catalyze its reaction
are
shown as purple spheres
Most enzymes are proteins, although some catalyc
RNA molecules have been identified. Enzymes
adopt a specific three-dimensional structure,
and
may employ organic (e.g. biotin, vitamin) and
inorganic (e.g.magnesium ion, zinc ion)
cofactors to assist in catalysis.
In 1897, Eduard Bucher submitted his first paper on
the ability of yeast extracts that lacked any living
yeast cells to ferment sugar. He named the enzyme
that brought about the fermentation of sucrose
“zymase".
In 1907, he received the Nobel Prize in Chemistry
"for his biochemical research and his discovery of
cell-
free fermentation"
Coenzymes – non-protein, organic, low molecular weight and
dialysable substance assosiated with enzime function.
The enzyme protein not always adequate to bring about
catalytic activity, this require certain non-protein small
additional factors, collectively referred to as cofactors for
catalysis.
The functional enzyme is referred to as holoenzyme
which is made up of protein part (apoenzyme) and non-
protein
part (coenzyme).
The term prosthetic group is used when a non-protein moiety
is tightly bound to the enzyme.
The term enzyme activator is referred to inorganic cofactor
(like Ca2+, Mg2+, Mn2+, Zn2+, Co2+, Cu2+ etc)
 Coenzymes – undergo alteration during the enzymatic
reactions, which are later regenerated
Most of the coenzymes are the derivatives of water soluble B-
complex vitamins.
There ate non-vitamin coenzymes:ATP (adenosine
triphosphat), CDP (cytidine diphosphate), UDP (uridine
diphosphate), SAM (S-adenosylmethionin or [active
methionine]), PAPS (phosphoadenosine phosphosulphate
[active sulfate]) etc.
There are althought protein coenzymes (thioredoxine is a
protein that serves a coenzyme for ribonucleotide
reductase
enzyme.
Coenzymes do not decide enzyme specificity.
Non-protein enzymes
 The common idea:
The higher the concentration of the substrate molecules, the
greater will be the rate of reaction.

But it does not work always…

some activation energy is necessary

i.e. – the energy is required by the reactants to undergo the
reaction
 Mechanism of enzyme catalysis:

1.Acid-base catalysis (importance of pH)

2.Substrate strain
3.Covalent catalysis
4.Entropy effect
5.Proximity catalysis
Substrates need a potential energy to reach a transition state, which
then decays into products. The enzyme stabilizes the transition
state,
reducing the energy needed to form products.

The energy levels of chemical reactions stages.

The chemical (biochemical) compounds which
take part in enzymatic reaction are:

Substrate [S]
Enzyme [E]
and, as a result of reaction, we get a

Product [P].
 Mechanism for a single substrate enzyme catalyzed reaction we
can show as follows: The enzyme (E) binds a substrate (S) and
produces a product (P).

Here k1, k–1 and k2 (sometime k1, k2 and k3,

or kf1, kr1 and kf2, kr2 ) represents a
velocity constants for the respective
reactions, as indicated by arrows
Change in concentrations over time for
enzyme [E], substrate [S], complex [ES]
and product [P]
Burst phase of enzymatic reaction
 Progress curve for an enzyme reaction.
The slope in the initial rate period is the initial rate of
reaction v. There is Michaele-Menten equation
which describes how this slope varies with the
concentration of substrate.
 Michaelis–Menten (Leon Michaelis and Canadian physician Maud Menten)
constant (or Brig’s and Haldane’s constant) is
defined as the substrate concentration (expressed
in moles/l) to produce half-maximum velocity
The Michaelis constant (Km) [or Brig’s and Haldane’s constant] is
given by formula:

Km = (k2 +k3)/k1

Sum of [ES] complex degradation velocity

constants k2 and k3 divided by [ES] complex
synthesis
Michaelis-Menten saturation curve
of an enzyme reaction
 Michaelis-Menten saturation curve
of an enzyme reaction
(an example curve with parametersVmax = 3.4 and Km = 0.4)
Michaelis–Menten kinetics is one of the best-known models
of enzyme kinetics. It is named after German biochemist
Leon Michaelis and Canadian physician Maud Menten.
The model takes the form of an equation describing the
rate of enzymatic reactions, by relating reaction rate to,
the concentration of a substrate S. Its formula is given by

Here, represents the maximum rate achieved by the

system, at maximum (saturating) substrate
concentrations. The
Michaelis constant (Km) is the substrate concentration at
which the reaction rate is half of . Biochemical reactions
involving a single substrate are often assumed to follow
Michaelis–Menten kinetics, without regard to the model's
 by taking the reciprocal
of both sides of Michaelis-Menten equation –
we get next equation
y = a x +
b

this equation gives us a linear plot

y = ax + b
named Lineweaver-Burk plot
 This Lineweaver-Burk plot known also as
Hanes-Woolf plot

or double-reciprocal plot of enzyme kinetics: here 1/V

is plotted as function of 1/[S]. The slope is Km/Vmax,
the intersect on the vertical axis is 1/ Vmax, and the
intersect on the horizontal axis is –1/Km.
This Lineweaver-Burk plot with inhibitor
action

changing of the slope due to inhibitor

There are factors affective enzyme activity:

1.Concentration of enzyme
2. Concentration of substrate
3.Effect of temperature
(bell-shaped curve)

4.Effect of pH
(bell-shaped curve)

5.Effect of products concentration

6.Effect of activators
7.Effect of time
8.Effect of light and radiation
 But the questions are:
Where in real life we can see the enzyme
action?
Where and how we can apply it?

And there are lot of examples:

Amylases from fungi and plants can produce
sugars from starch. In baking, amylases
catalyze breakdown of starch in the frour to
sugar.

Yeast fermentation of sugar produces the carbon

dioxide that raises the dough.
Similar enzyms produce alcohol in brewery.
there although can be an Inhibition Reaction
Types of
Inhibition
Reaction
Lineweaver–Burk plots
show us the different
types of
reversible enzyme inhibitors.

The arrow on plot shows the effect of

increasing concentrations of
inhibitor.
Regulation of enzymes activity in the living system

1.Allosteric regulation
2.Activation of latent enzymes
3.Compartmentation of metabolic
pathways 4.Control of enzymes synthesis
5.Enzyme
degradation
6.Isoenzymes
 UNITS OF ENZYME ACTIVITY
(Enzyme Commission of IUB)
One katal (abbreviated – kat) denotes the conversion
of one mole substrate per second (mol/sek)

SI (System Intrenational) Unit

One SI unit provides conversation of one micromol
of
substrate per minute.
1 IU = 16,67 nkat (nanokatal)
1 kat = 6×107 IU
 There are thousand kind of enzymes
and variants of their action in biological objects…

…so, we need somehow to systemize them…

And there is a classification of

enzymes:
 ENZYMES

1 OXIDOREDUCTASES: catalyze
oxidation/reduction reactions
2 TRANSFERASES: transfer a functional
grout (e.g. a methyl or phosphate group)
3 HYDROLASES: catalyze the hydrolysis of
various bonds
4 LYASES: cleave various bonds by means other than
hydrolysis and oxidation
5 ISOMERASES : catalyze isomerisation
changes within a single molecule
6 LIGASES: join two molecules with covalent
bonds.
Classification of enzymes
 Oxidoreductase
an enzyme that catalyzed this reaction would be an
oxidoreductase:
A– + B → A + B –
In this example, A is the reductant (electron donor) and B is the
oxidant (electron acceptor).

In biochemical reactions, the redox reactions are sometimes

more difficult to see, such as this reaction from
glycolysis:
Pi + glyceraldehyde-3-phosphate + NAD+ → NADH + H+ +
1,3-
bisphosphoglycerate
In this reaction, NAD+ (nicotin-amid-dinucleotid) is the oxidant
 Oxidoreductase
Oxidoreductases are classified as EC 1 in the EC number classification of
enzymes. Oxidoreductases can be further classified into 22
subclasses:
EC 1.1 includes oxidoreductases that act on the CH-OH group of donors (alcohol
oxidoreductases) EC 1.2 includes oxidoreductases that act on the aldehyde or oxo group of
donors
EC 1.3 includes oxidoreductases that act on the CH-CH group of donors (CH-CH oxidoreductases)
EC 1.4 includes oxidoreductases that act on the CH-NH2 group of donors (Amino acid oxidoreductases,
Monoamine oxidase)
EC 1.5 includes oxidoreductases that act on CH-NH group of
donors EC 1.6 includes oxidoreductases that act on NADH or
NADPH
EC 1.7 includes oxidoreductases that act on other nitrogenous compounds as
donors EC 1.8 includes oxidoreductases that act on a sulfur group of donors
EC 1.9 includes oxidoreductases that act on a heme group of donors
EC 1.10 includes oxidoreductases that act on diphenols and related substances as
donors EC 1.11 includes oxidoreductases that act on oeroxide as an acceptor
(peroxidases)
EC 1.12 includes oxidoreductases that act on hydrogen as donors
EC 1.13 includes oxidoreductases that act on single donors with incorporation of molecular oxygen
(oxygenases) EC 1.14 includes oxidoreductases that act on paired donors with incorporation of molecular
oxygen
EC 1.15 includes oxidoreductases that act on superoxide radicals as acceptors
EC 1.16 includes oxidoreductases that oxidize metal ions
EC 1.17 includes oxidoreductases that act on CH or CH2
groups
EC 1.18 includes oxidoreductases that act on iron-sulfur proteins as donors
 Transferase

transferase is the general name for the class of enzymes that enact the transfer of
specific functionsl groups (e.g. a methyl or glycosyl group) from one molecule (called
the donor) to another (called the acceptor). They are involved in hundreds of different
biochemical pathways throughout biology, and are integral to some of life’s most
important processes.
Transferases are involved in a myriad of reactions in the cell. Some examples of these
reactions include the activity of CoA transferase, which transfers thiol esters, the
action of N-acetyltransferase is part of the pathway that metabolizes tryptophan, and
also includes the regulation of PDH, which converts pyruvate to Acetyl
CoA. Transferases are also utilized during translation. In this case, an amino acid
chain is the functional group transferred by a Peptidyl transferase. The transfer
involves the removal of the growing amono acid chain from the tRNA molecule in the
A-site of the ribosome and its subsequent addition to the amino acid attached to the
tRNA in the P-site.
Mechanistically, an enzyme that catalyzed the following reaction would be a transferase:
In the above reaction, X would be the donor, and Y would be the acceptor. "Group"
would be the functional group transferred as a result of transferase activity. The
donor is often a coenzyme.
 Transferase
Transferases are classified as EC 2 in the EC number classification of
enzymes. Transferases can be further classified into 10
subclasses:

EC 2.1.Single carbone transferases

EC 2.2. Aldehyde and keton transferases
EC 2.3.Acyl transferases
EC 2.4. Glycosil, hexosyl and pentosyl
transferases EC 2.5.Alkyl and aryl transferases
EC 2.6.Nitrogenous
transferases EC 2.7.Phosphorus
transferases EC 2.8.Sulfur
transferases
EC 2.9.Selenium
transferases EC 2.10.Metal
transferases
 Hydrolase

a hydrolase is an enzyme that catalyzes the hydrolysis of a

chemocal bond. For example, an enzyme that catalyzed the
following reaction is a hydrolase:
A–B + H2O → A–OH + B–H
 Hydrolase
Hydrolases are classified as EC 3 in the EC numbers classification of enzymes.
Hydrolases can be further classified into several subclasses, based upon the bonds
they act upon:

EC 3.1ester bonds (esterases: nucleases, phosphodiesterases, lipase, phosphatase)

EC 3.2.sugars (DNA glycosydases, glycoside hydrolases)
EC 3.3.eter bonds
EC 3.4.peptide bonds (proteases/peptidases)
EC 3.4.carbon-nitrogen bonds, other than peptide bonds
EC 3.6.acid anhydrides (acid anhydride hydrolases, including helicases and
GTPase) EC 3.7.carbon-carbon bonds
EC 3.8.halide bonds
EC 3.9.phosphorus-nitrogen bonds
EC 3.10.sulphur-nitrogen bonds
EC 3.11.carbon-phosphorus
bonds EC 3.12.sulfur-sulfur
bonds
EC 3.13.cfrbon-sulfur bonds
 Lyase

lyase is an enzyme that catalyzes the breaking (an “elimination”

reaction) of various chemical bonds by means other than hydrolisis
(a “susstitution” reaction) and oxydation, often forming a new
double
bond or a new ring structure.
The reverse reaction is also possible (called a “Michael addition”).
For example, an enzyme that catalyzed this reaction would be a
lyase: ATP → cATP + PPi
Lyases differ from other enzymes in that they require only one
substrate for the reaction in one direction, but two substrates for
the
reverse reaction.
 Lyase

Lyases are classified as EC 4 in the EC number classification of enzymes. Lyases can

be further classified into seven subclasses:

EC4.1.includes lyases that cleave carbon-carbon bonds, such

as Decarboxylases (EC 4.1.1),
Aldehyde lyases (EC 4.1.2),
Oxo acid lyases(EC 4.1.3)
and others (EC 4.1.99)
EC 4.2.includes lyases that cleave carbon-oxygen bonds, such as dehydratases
EC 4.3.includes lyases that cleave carbon-nitrogen bonds
EC 4.4.includes lyases that cleave carbon-sulfur bonds
EC 4.5.includes lyases that cleave carbon-halide
bonds
EC 4.6.includes lyases that cleave phosphorus-oxygen bonds, such as
adenilate
cyclase and guanilat cyclase
EC 4.99.includes other lyases, such as ferrochelase
 Isomerase

Isomerases are a general class of enzymes which convert a

molecule from one isomer to another. Isomerases can either
facilitate
intramolecular rearrangements in which bonds are broken and formed
or they can catalyze conformational changes. The general form of
such a reaction is as follows:
A–B → B–A
There is only one substrate yielding one product. This product has
the same molecula formula as the substrate but differs in bond
connectivity or spatial arrangements. Isomerases catalyze reactions
across many biological processes, such as in glycolysis and
carbohydrate metabolism.
 Isomerase
Isomerase are classified as EC 5 in the EC number classification of
enzymes.
Isomerase can be further classified into seven subclasses:

1.racemases, epimerases
2.cis-trans isomerases
5.3.intramolecular
oxidoreductases
5.4.intramolecular transferases
5.5.intramolecular lyases
 Ligase
ligase (from the Latin verb ligāre — "to bind" or "to glue together") is an
enzyme that can catalyze the joining of two large molecules by forming a
new chemical bond, usually with accompanying hydrolysis of a small
chemical group dependent to one of the larger molecules or the enzyme
catalyzing the linking together of two compounds, e.g., enzymes that
catalyze joining of C-O, C-S, C-N, etc. In general, a ligase catalyzes the
following reaction:
Ab + C → A–C + b
or sometimes
Ab + cD → A–D + b + c
where the lowercase letters signify the small,
dependent groups. Ligase can join
two complementary fragments of nucleic acid and repair single stranded
breaks that arise in double stranded DNA during replication.
 Ligase
Ligases are classified as EC 6 in the EC number classification of enzymes.
Ligases can be further classified into six subclasses:

EC 6.1.includes ligases used to form carbon-oxygen

bonds EC 6.2.includes ligases used to form carbon-sulfur
bonds
EC 6.3.includes ligases used to form carbon-nitrogen bonds
(including
argininosuccinate synthetase)
EC 6.4.includes ligases used to form carbon-carbon bonds
EC 6.5.includes ligases used to form phosphoric ester
bonds EC 6.6.includes ligases used to form nitrogen-metal
bonds
Thank You for attention…

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