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METHODICAL POINTING
from the course
“Biological and bioorganic chemistry”
(part 7. Enzymes as biological catalysts)
Readers:
Kyiv-2018
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CONTENT
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7. Enzymes as natural biological catalysts
Structure of enzymes
Enzymes
Apoenzyme (protein
part) Cofactor
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.
• ubiquinone:
• carnitine:
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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
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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• 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
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. 7.7. The dependence of the rate of enzymatic reaction on the pH of medium
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
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.
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
7.9.1. Oxidoreductases
donor : acceptor-oxidoreductase.
7.9.2. Transferases
7.9.3. Hydrolases
R - X + HOH → ROH + HX
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:
These enzymes form C-C, C-N, C-O, C-S bonds. There are 5 subclasses of
ligases.
7.9.6. Isomerases
• cis-trans-isomerases.
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).
For example:
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
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
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
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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
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.
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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
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.
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Substrate
Active site
Enzyme Enzyme-substrate
complex
(a) (b)
In this model:
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)
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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;
• 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.
Comparison of Lock and key and Induced fit models is gien on Fig. 7.12.
Fischer: Lock
and key
Koshland:
Induced fit
Fig. 7.12. Comparison of Lock and key and Induced fit models
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7.13. Enzymatic reaction steps
E + S ES
Within the active site of the ES complex, the reaction occurs to convert
substrate to product (P):
ES → E + P
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
5 Products are
released. 4 Substrates are
converted into
products.
Products
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
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.
Active
site
Competitive
inhibitor
Enzyme
Noncompetitive
inhibitor
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:
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Non- Phosphorylated
phosphorylated Inactive
protein Active
protein
Other enzymes are active in
dephosphorylated state and
phosphorylation will inactivate them