Enzymes

1. BIOCHEMISTRY
ENZYMES

2. 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.

3. Enzyme (human glucolase)
Zinc ions that are needed for the enzyme to catalyze its reaction are
shown as purple spheres

4. 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"

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6. 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)

7. 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.
Specificity is mostly dependent on the apoenzyme.

8. Non-protein enzymes

9. 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

10. Mechanism of enzyme catalysis:
1.Acid-base catalysis (importance of pH)
2.Substrate strain
3.Covalent catalysis
4.Entropy effect
5.Proximity catalysis

11. Substrates need a potential energy to reach a transition state, whichthen
decays into products. The enzyme stabilizes the transition state,
reducing the energy needed to form products.
The energy levels of chemical reactions stages.

12. 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].

13. 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

14. Change in concentrations over time for
enzyme [E], substrate [S], complex [ES]
and product [P]

15. Burst phase of enzymatic reaction

16. 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.

17. 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
velocity constant k1

18. Michaelis-Menten saturation curve
of an enzyme reaction

19. Michaelis-Menten saturation curve
of an enzyme reaction
(an example curve with parametersVmax = 3.4 and Km =0.4)

20. 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
underlying assumptions.

21. this equation gives us a linear plot
y = ax + b
named Lineweaver-Burk plot
by taking the reciprocal
of both sides of Michaelis-Menten equation –
we get next equation
y = a x + b

22. 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.

23. This Lineweaver-Burk plot with inhibitor action
changing of the slope due to inhibitor action

24. 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

25. 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:

26. 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.

27. there although can be an Inhibition Reaction

28. Types of
Inhibition
Reaction

29. Lineweaver–Burk plots show
us the different types of
reversible enzyme inhibitors.
The arrow on plot shows the effect of increasing
concentrations of inhibitor.

30. 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

31. 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

32. 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:

33. 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.

34. Classification of enzymes

35. 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
(electron acceptor), and glyceraldehyde-3-phosphate is the
reductant (electron donor).

36. 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 (alcoholoxidoreductases)
EC 1.2includes 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-CHoxidoreductases)
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 ofdonors
EC 1.6 includes oxidoreductases that act on NADH orNADPH
EC 1.7 includes oxidoreductases that act on other nitrogenous compounds as donors
EC 1.8 includes oxidoreductases that act on a sulfur group ofdonors
EC 1.9 includes oxidoreductases that act on a heme group ofdonors
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 asdonors
EC 1.13includes 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 asacceptors
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
EC 1.19 includes oxidoreductases that act on reduced flavodoxin as a donor
EC 1.20 includes oxidoreductases that act on phosphorus (P) or arsenic (As) in donors
EC 1.21 includes oxidoreductases that act on X-Hand Y-H to form an X-Y bond
EC 1.97 includes otheroxidoreductases

37. 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.

38. Transferase
Transferases are classified as EC 2 in the EC number classificationof
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

39. 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

40. 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, glycosidehydrolases)
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

41. 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.

42. 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, suchas
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-sulfurbonds
EC 4.5.includes lyases that cleave carbon-halide bonds
EC 4.6.includes lyases that cleave phosphorus-oxygen bonds, such asadenilate
cyclase and guanilat cyclase
EC 4.99.includes other lyases, such as ferrochelase

43. 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.

44. Isomerase
Isomerase are classified as EC 5 in the EC number classification ofenzymes.
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

45. 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.

46. 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-oxygenbonds
EC 6.2.includes ligases used to form carbon-sulfurbonds
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

47. Thank You for attention…
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