1. Enzymes II
Konstantin D. Malyi
Department of Biochemistry
Crimean Medical Academy
Simferopol, 24.09.2021
2. Structural and functional
describing of enzymes
• The word enzyme comes from Greek "in leaven". As early as the late
1700s and early 1800s the digestion of meat by stomach secretions
and the conversion of starch to sugars by plant extracts and saliva
were observed.
• Studying the fermentation of sugar to alcohol by yeast, Louis Pasteur
came to the conclusion that this fermentation was catalyzed by
“ferments" in the yeast, which were thought only to function in the
presence of living organisms.
• In 1897, Hans and Eduard Buchner inadvertently used yeast extracts
to ferment sugar despite the absence of living yeast cells.
The term "enzyme" was used to describe the substance(s) in yeast
extract which brought about the fermentation of sucrose.
3. Two variants of the names of enzymes – actually
enzymes, and ferments.
More often the term of “ferments” is used to refer to
the fermentation processes.
The fermentation process usually refers to the use of
microorganisms in food production, in industry,
when describing the metabolism of carbohydrates in
the cell…
A common feature of all enzymes is the ability to
catalyze. Enzymes are biological catalysts.
4. Catalysis – acceleration of rate of chemical
reaction by interaction of catalyst with
reactants, across creation of intermediate
complex, with lowering of free energy of
transient state reaction, and without
consumption of catalyst.
6. Units of enzyme activity
International unit (symbol U, sometimes also IU) –
amount of substrate reacting or product produced
per minute, usually
micromoles/min, mmol/min
SI = katal
amount (mol) of substrate reacting or product
produced per second, mol/sec
9. Nomenclature of enzymes
Most enzymes are named according to the function or substrate that they
carry out with the appended suffix “-ase”.
- DNA polymerase catalyzes the polymerization of deoxynucleotides to
form DNA.
- Lipoyl transferase catalyzes the transfer of the lipoyl moiety from one
protein to another.
- Hexokinase, catalyzes the phosphorylation of glycose by transfer of
phosphoryl group from ATP.
- Cysteine desulfurase catalyzes the liberation of sulfur from the amino
acid cysteine.
Some enzymes that have been known since before a systematic means of
nomenclature arose are still called by their trivial (historical) names,
such as trypsin, pepsin, chymotrypsin, etc.
Still, a numeric system of nomenclature has been established.
10. The Enzyme Commission Number (EC Number)
is a numerical classification scheme for enzymes
11. Enzyme Comission number of enzymes
The Enzyme Commission Number (EC Number) is a numerical classification
scheme for enzymes, based on the chemical reactions they catalyze. The chemical
reaction catalyzed is the specific property that distinguishes one enzyme from
another. EC numbers specify enzyme-catalysed reactions.
For example the enzyme hexokinase catalyzes the transfer of a phosphoryl group from ATP to
glucose. It’s Enzyme Commision number (EC number) is 2.7.1.1
– The first digit denotes the class name (transferase)
– The second digit denotes the subclass (phosphotransferase)
– The third digit denotes the acceptor atom (hydroxyl group)
– The fourth digit denotes the acceptor (D-glucose)
ATP + D-glucose ADP + D-glucose 6-phosphate
12. EC functional classification of enzymes
• I Oxidoreductases Transfer of electrons (hydride ions or H atoms)
• II. Transferases Group transfer reactions
• III. Hydrolases Hydrolysis reactions (transfer of functional
groups to water)
• IV. Lyases Addition of groups to double bonds or
formation of double bonds by removal of groups
• V. Isomerases Transfer of groups within molecules
to yield isomeric forms
• VI. Ligases Formation of C-C, C-S, C-O and C-N bonds by
condensation reactions coupled to ATP cleavage
• VII. Translocases Catalyse the movement of ions or molecules across
membranes or their separation within membranes
16. Biological role of apoenzyme - specificity and
effectiveness of enzyme.
Biological role of cofactor – catalytic reaction.
Many vitamins, for example group B vitamins, are
precursors of enzyme cofactors, without which an
enzymatic reaction is impossible, which leads to a clinical
picture of vitamin deficiency – avitaminosis (“acquired
enzymopathy”).
23. Functional sites of enzymes
• Active site (may be divided on site of
catalysis and site of specifity)
• Allosteric site
• Site of self-association (assemling)
• Mounting site (interaction with membrane,
anchoring)
• Antigen site
32. Mechanisms of substrate binding
Key and lock theory (Fisher)
Interaction between substrate and active site
describe as rigid “key and lock” analogy
Induced fit theory (Koshland)
Substrate induced conformational change in
enzyme molecule.
33.
34. Specific interaction of enzyme with substrate
Fischer's lock and key hypothesis . Koshland's induced fit hypothesis
of enzyme action of enzyme action
35. aA + bB cC + dD
V dir = K dir [A]a [B]b
Vrev = Krev [C]c [D]d
36. aA + bB cC + dD
V dir = K dir [A]a [B]b
Vrev = Krev [C]c [D]d
Vdir = Vrev
K dir [C]c [D]d
Keq = --- = ------------
Krev [A]a [B]b
DG = -RT ln Keq
37. Progress of the reaction
Products
Reactants
∆G < O
EA
D
C
B
A
D
D
C
C
B
B
A
A
The Activation Energy Barrier
The initial
energy needed
to start a
chemical
reaction is
called the
activation
energy (EA)
often supplied
in the form of
heat from the
surroundings
Transition state
EA with
enzyme
is lower
Course of
reaction
with enzyme
• Chemical reactions
involves bond breaking
and bond forming
AB + CD AC + BD
-DH
-DG
How do
Enzymes
Catalyze
reactions ?
by lowering
the EA
Barrier
38. Physical effects of enzyme action:
- Effect of straining (creation of intermediate
complex, where molecule(s) of substrate
will be in strain conformation, and new
bonds will be more preferential
- Effect of orientation, when enzyme bound
molecules of substrate and orientate it
optimally for interaction
39. Catalysis may be heterogeneous or homogeneous, the
latter is the case with enzymes. Enzymes employ
many modes of chemical catalysis:
• electrophilic including metal ion catalysis,
• Nucleophilic,
• Acid-base
• Special effects by cofactors, within which are
electrostatic, steric, H-bonding and differential
solvation effects.
A basic form of rate acceleration, also used by
enzymes, is intramolecular catalysis.
40. Mechanisms of enzyme action
Enzyme is not simply a template to which the substrates bind for ready
reaction, but that groups on its surface - hydrophilic groups for sure -
participate in catalysis - chemical catalysis by groups on the enzyme
Amount of chemical participation varies widely among enzymes
Very often the transition state of a reaction involves charge separation,
which is energetically unfavorable. Any participation by a catalyst
which spreads the charge more widely thus stabilizes the transition
state and lowers the energy of activation.
41.
42. Serine proteases
Serine proteases are a class of proteolytic enzymes whose
catalytic mechanism is based on an active-site serine residue
Trypsin, chymotrypsin, elastase.
Degradative proteases of the digestive system.
Plasmin, tissue plasminogen activator, thrombin.
Regulatory proteases, found in amplification cascades associated
with blood clotting (thrombogenesis) or the dissolving of blood clots
(thrombolysis) - opposing processes that together regulate hemostasis
Kallikreins.
Regulatory proteases that function to activate peptide pro-hormones
and growth factors by cleaving pro-sequences from the zymogen forms of such
peptides
Subtilisin.
A degradative bacterial protease, sometimes added to laundry detergents to
break down protein-pigment complexes in blood and grass stains
43. Catalytic mechanism of serine proteases
Three key amino acid radicals in
active site:
serine, aspartic acid and
histidine.
• Ser is the actual nucleophile attacking the
substrate
• His functions as either an acid or base to
facilitate the catalytic steps
• Asp functions to perturb the pKa of the His
to permit the acid/base property of the His.
1.
44. Catalytic mechanism of serine proteases
Three key amino acid radicals in
active site:
serine, aspartic acid and
histidine.
• Ser is the actual nucleophile attacking the
substrate
• His functions as either an acid or base to
facilitate the catalytic steps
• Asp functions to perturb the pKa of the His
to permit the acid/base property of the His.
1.
49. Influence of substrate concentration
on rate of enzyme reaction
Equation of Michaelis-Menten
Vmax [S]
V = -------------;
Km + [S]
Vmax
when V = --------
2
Km = [S],
50. Influence of substrate concentration
on rate of enzyme reaction
Equation of Michaelis-Menten
Vmax [S]
V = -------------;
Km + [S]
Vmax
when V = --------
2
Km = [S],
54. Regulation
of enzyme activity
• Activators
- Nonspecific (temperature, pH, salt concentration)
- Specific (cofactors, coregulators, allosteric regulators)
• Inhibitors
- nonspecific
- specific (competitive, noncompetitive. uncompetitive)
55. Functional sites of enzymes
• Active site (may be divided on site of
catalysis and site of specifity)
• Allosteric site
• Site of self-association (assemling)
• Mounting site (interaction with membrane,
anchoring)
• Antigen site
61. Regulation of enzyme activity
Factor of
regulation
Effector Result Time of answer
Substrate
availability
Substrate Change in velocity Immediately
Product inhibition Product Change Vmax
and/or Km
Immediately
Allosteric control End product Change Vmax
and/or Km
Immediately
Covalent
modification
Another enzyme Change Vmax
and/or Km
Up to minutes
Synthesis or
degradation of
enzyme
Hormones,
metabolites
Change of the
anount af the
enzyme
Hours to days
