biocatalysts - uniba.sk
TRANSCRIPT
ENZYMES BIOCATALYSTS
2009/2010
INGRID ŽITŇANOVÁ
ENZYMOLOGY
HISTORY
17th -18th century – digestion of meat caused by stomach secretions
- conversion of starch to glucose by salivaLouis Pasteur
Eduard Buchner
19th century – L. Pasteur – fermentation of sugar to alcohol by yeasts
- vital force in yeasts required for this fermentation
1897 – Eduard Buchner – ability of yeast extracts that lacked
any living yeast cells to ferment sugar – in 1907 – Nobel
Prize for chemistry - discovery of cell free fermentation
1926 – James B. Sumner isolated the first enzyme – urease and
prooved its protein character
en zyme – in yeasts
• Enzymes are biocatalysts
✓ Increase the rate of a reaction
✓ Not consumed by the reaction
✓ Enzymes are often very “specific” – promote only 1 particular
reaction
✓ In the single cell - more than 3000 enzymes
BIOCATALYSTS VS. INORGANIC
CATALYSTS
Enzymes (biocatalysts):
1) More efficient - higher reaction rate
2) Milder reaction conditions (20 - 40°C, pressure 0.1 MPa, pH = 7)
3) Higher specificity of the reaction
4) Ability to be regulated at different levels (inhibitors, activators)
5) They are non-toxic
6) Enzymes – organic compounds, chemic. catalysts – inorg. compounds
Catalyst rate enhancement
Inorganic catalysts 102 -104 fold
Enzymes up to1020 fold
Catalyst time for reaction
With enzyme 1 second
Without enzyme 3 x 1012 years
How much is 1020 fold?
ENZYME STRUCTURE
• Enzymes are proteins (chain of amino acids)
• Enzyme will twist and fold into a specific shape due
to how the amino acids are attracted to each other
• Enzyme shape attracts specific
molecules - substrates – molecules
that bind to the enzyme
Carbonic anhydrase
in lungs: H2CO3 CO2 + H2O
➢ Enzymes DO NOT change the equilibrium constant
of a reaction (accelerate the rate of the forward and
reverse reactions equally)
Carbonic anhydrase
in tissues: CO2 + H2O H2CO3
Carbonic anhydrase
CO2 + H2O H2CO3
ENZYMES
SIMPLE COMPLEX (HOLOENZYME)
APOENZYMES
(Protein)
COFACTOR (Nonprotein)
COENZYME(loosely bound)
PROSTHETIC GROUP
(tightly bound)
ORGANIC INORGANIC
HOLOENZYME
Inorganic elements serving as enzyme cofactors
Cytochrome oxidase
Cytochrome oxidase, catalase, peroxidase
Pyruvate kinase
Hexokinase, pyruvate kinase
Arginase,
Dinitrogenase
Urease
Glutathione peroxidase
Carboanhydrase, alcohol dehydrogenase
Cu2+, Zn2+, Mn2+ Superoxide dismutase
Cofactors
◼ cofactors can serve several apoenzymes:
NAD+ (nicotinamide adenine dinucleotide) - a cofactor for a great
number of dehydrogenases: alcohol dehydrogenase,
malate dehydrogenase
lactate dehydrogenase reactions
Role of organic cofactors:
transport of chem. groups from 1 reactant to another
Classification of cofactors according to
the type of a transferred molecule
1) Transfer of H atoms
NAD+ (nicotine amide adenine dinucleotide) - transport of H-
FAD (flavine adenine dinucleotide) – transport of 2H
FMN (flavine mononucleotide), lipoic acid - transport of 2H
3) Transfer of groups of atoms
adenosine phosphates (ATP, ADP) - phosphate group
coenzyme A – acyl groups
thiamine diphosphate - aldehydes
pyridoxal phosphate – amine groups
biocytin – CO2
tetrahydrofolate (coenzyme F) – one-carbon groups
2) Transfer of electrons
coenzyme Q, porfyrin derivatives
Vitamins are often converted to coenzymes
Vitamin Coenzyme Function
Thiamin diphosphate decarboxylation
Flavin mononucleotide (FMN) carries hydrogen
Nicotinamide adenine dinucleotide carries hydrogen H-
(NAD+), (NADH)
B1
B2
B3
B5
pantothenic acid
H (B7) Biocytin CO2 fixation
Coenzyme A acyl group carrier
B9-folic acid
B12-cobalamine
Tetrahydrofolate carries one carbon units
Methylcobalamine, adenosylcobalamine
ACTIVE SITE
Active site
Substrate
ACTIVE SITE
▪ACTIVE SITE = pocket in the enzyme where substrates
bind and catalytic reaction occur
CATALYTIC SITE(where the reaction proceeds)
BINDING SITE(where a substrate binds)
▪ Some enzymes contain more active sites (2 - 4), they can
bind more substrate molecules
▪ Aminoacids of the active site can be located at different
regions of a polypeptide chain
Aminoacids of the active site can be located at different regions of a polypeptide chain
➢ Substrates bind in active site by following interactions:
➢ hydrogen bonds
➢ hydrophobic interactions
➢ ionic interactions
➢ covalent bonds (occasionally)
➢ The interactions hold the substrate in the proper orientation for most
effective catalysis
➢ The ENERGY derived from these interactions = “Binding energy“
hydrogen
bonding
binding pocketionic interaction
ionic interaction
hydrophobic
interaction
2. non-covalent interactions
between substrate and
the active site:
- hydrogen bonding
- ionic interactions
- hydrophobic interactions
Interactions between
enzyme and substrate
1/ E + S E-S Formation of E-S complex
2/ E-S E-S* Activation of the complex
3/ E-S* E-P Conversion of substrate to a product
4/ E-P E + P Separation of product from enzyme
ES* = enzyme/transition state complex
Stages of enzyme reaction
E + S ES
First step of enzyme catalysis
FORMATION OF THE ENZYME-SUBSTRATE
COMPLEX
ES transition state complex
Second step
FORMATION OF THE TRANSITION
STATE COMPLEXNote change
Transition State:
a. Old bonds break and
new ones form.
b. Substance is neither
substrate nor product
c. Unstable short lived
species with an equal
probability of going
forward or backward.
Third step
FORMATION OF THE ENZYME-PRODUCT
COMPLEX
ES* EP
Fourth step
RELEASE OF THE PRODUCT
EP E + P
Mechanisms of substrate conversion
• Enzyme binds
2 substrates,
that they are in
close vicinity
• Charges in the
active site
induce changes
in the charges in
S molecule
• Deformation of S
facilitates its
conversion to a
product
• Activation energy is the energy required to start a
reaction.
MECHANISM OF ENZYME ACTION
• Enzymes decrease the activation energy of a reaction
by formation of the active enzyme - substrate complex
Uncatalyzed reaction
Catalyzed reaction
Substrate
Product
En
erg
y
Transition state
• The lower the free energy of activation, the more molecules have
sufficient energy to pass through the transition state, and, thus,
the faster the rate of the reaction.
Enzyme activity
The katal (symbol: kat) - the SI unit of catalytic activity
1 kat = mol . s-1
One katal is the catalytic activity that changes one mole
of substrate per second at optimal pH.
Enzyme
Substrate Product
◼ SPECIFIC ACTIVITY – katal/kg (μkat/mg) protein
◼ MOLAR ACTIVITY – katal/mol protein
1 U = μmol . min-1
1 kat = mol/s = 60 mol/min= 60.106 μmol.min-1 = 6.107 U
1 U = μmol.min-1 = 10-6 mol/60 s = 16.7 . 10-9 kat
Enzyme has SPECIFICITY – it can discriminate among
possible substrate molecules:
ENZYME SPECIFICITY
EnzymeEnzyme
SubstrateSSS
Enzymes are very specific
and only work with certain substrates
SUBSTRATE SPECIFICITY(apoenzyme responsible)
1) Strictly specific enzymes - only react with a single
substrate (DNA polymerase, urease)
2) Less specific enzymes
a. Group specific - recognize a functional group (-OH, -NH2...)
(alcoholdehydrogenase - converts methanol, ethanol,
ethylene glycol)
b. Linkage specific – particular type of chemical bond regardless
. of the rest of the molecular structure (peptidase, esterase)
SPECIFICITY OF EFFECT (cofactor responsible)
OXIDOREDUCTASES – oxidation/reduction reactions - transfer of H and O atoms or electrons from one substance to another (alcoholdehydrogenase)
TRANSFERASES – transfer of a functional group - methyl-, acyl-, amino- or
phosphate group (hexokinase)
HYDROLASES – catalyze hydrolysis of various bonds (carboxypeptidase A)
LYASES – cleave bonds by means other than hydrolysis and oxidation
(pyruvate decarboxylase)
ISOMERASES – intramolecular changes of „S“ (maleate isomerase)
LIGASES – join two molecules with covalent bonds with the
use of energy from ATP (pyruvate carboxylase)
MODELS FOR ENZYME/SUBSTRATE
INTERACTIONS
1) Lock and Key Model (Emil Fischer 1894)
➢ This model assumed that only a substrate of a proper shape
could fit with the enzyme
Substrate
Active siteES complex
Enzyme
Enzyme
Substrate
A. Substrate (key) fits into a perfectly shaped space in the enzyme
(lock)
B. Highly stereospecific
C. Site is preformed and rigid
1) Lock and Key Model (Emil Fischer 1894)
2) Induced Fit Model (Daniel Koshland 1958)
➢ This model assumes continuous changes in the active site
structure as a substrate binds
Enzyme
Substrate
Active site
ES complex
◼ Takes into account the flexibility of proteins
◼ A substrate fits into a general shape in the enzyme, causing the enzyme to change shape (conformation)
◼ Change in protein configuration leads to a near perfect fit of
substrate with enzyme
Induced fit model
• Uncatalyzed reactions often are extremely slow.
Principles of Catalysis
• They are slow because of the heigh activation energy
• Enzymes lower the activation energy by creating an ES
(enzyme-substrate) complex which reduces bond strength in
the substrate and makes the substrate easier to convert to the
product.
Enzyme Nomenclature
1. Trivial names
2. Systematic nomenclature
Enzyme Nomenclature
1. Trivial names
◼ everyday use (pepsin, trypsin)
Usually named by suffix –ase to: - the name of a substrate (urease)
- the catalytic reaction (glucose
oxidase)
Some examples:
Alcohol dehydrogenase - oxidation of alcohols
DNA polymerase - polymerization of nucleotides
Protease - hydrolysis of proteins
Methyltransferase - methyl group transfer
2. Systematic names
◼ Introduced in 1961 (enzyme commision of IUB)
◼ Systematic names:
a) characterizing catalytic reaction
b) recommended – commonly used
c) international – code number
L-lactate + NAD+ pyruvate + NADH + H+
2. Systematic names
a) Characterizing the reaction:
L-lactate : NAD+ - oxidoreductase
name of substrates + name of the reaction catalyzed + suffix
(separated by the colon)
–ase
b) Recommended name: Lactate dehydrogenase
c) Code number : EC 1.1.1.1
◼ EC 1.x.x.x oxidoreductases
◼ EC 2.x.x.x transferases
◼ EC 3.x.x.x hydrolases
◼ EC 4.x.x.x lyases
◼ EC 5.x.x.x isomerases
◼ EC 6.x.x.x ligases (synthetases)
CODE NUMBERS OF ENZYMES
b) Recommended name: Lactate dehydrogenase
L-lactate + NAD+ pyruvate + NADH + H+
c) Code number : EC 1. 1. 1. 1
oxidoreductase
acting on the CH-OH group
NAD+ as acceptor
alcohol dehydrogenase
ISOZYMES – ISOENZYMES
• catalyze the same reaction
• have different primary structure
• are produced by different genes (= true isozymes), or produced
by different posttranslational modification (= isoforms)
• have different physical and chemical properties
• can be localized in different organs and cell compartments
pyruvate
Lactate dehydrogenase
LDH1 – LDH5
• Slightly different amino acid sequence
• Detection of specific LDH isozymes in the blood - diagnostics
of tissue damage such as occurs during myocardial infarction
lactate
Lactate dehydrogenase – composed of M a H subunits
5 isomers of lactate dehydrogenase
M4
M3H
M2H2
MH3
H4
M4 M3H M2H2 MH3 H4
Liver
Muscle
White cells
Brain
Red cells
Kidney
Heart
Separation by electrophoresisLDH-1
LDH-2
LDH-3
LDH-4
LDH-5
LDH1
LDH2
LDH5
Control serum
LDH1
LDH2
LDH3
LDH3
LDH5
Regulation of enzyme activity
A) Without the change in the quantity of enzyme
molecules 1) Physico-chemical factors
2) Presence of inhibitors and activators
3) Allosteric regulation of enzyme activity
4) Regulation by modification of enzyme molecule
5) Compartmentalization of enzymes
B) With the change of the number of enzyme
molecules1) Induction and repression
2) Regulated degradation of proteins
1. Physico-chemical factors
➢ Substrate concentration
➢ Temperature
➢ pH
➢ Ionic strength
➢ Redox potential
Substrate Concentration
• for isosteric enzymes
• for single-substrate reactions
Saturation curve
Km
½ Vmax
• fixed amount of enzyme
MICHAELIS and MENTEN equation
v - reaction rate
vmax - maximal reaction rate
[S] - substrate concentration (mol/L)
Km - Michaelis constant (mol/L)
vmax [S]
v =
Km + [S]
The MICHAELIS´ CONSTANT (Km) – is the substrate
concentration at which the reaction rate is half of maximal,
and is an inverse measure of the substrate's affinity for the
enzyme
Maud Menten
Leonor Michaelis
Lineweaver – Bürk equation
(reciprocal transformation of Michaelis -Menten equation)
1 Km + [S] Km 1 [S] Km 1 1
v vmax [S] vmax [S] vmax [S] vmax [S] vmax
=== + +. .
Vmax [S]
v =
Km + [S]
• It is valid for single substrate reactions
-1/Km
1/vmax
1/v
1 Km 1 1
v vmax [S] vmax
= +.
Lineweaver – Burk plot
1/S
y a x b
y = ax + b
Multi-substrate reactions
1) Ternary-complex mechanism
(sequential)
2) Ping-pong mechanism• Formation of binary complexes – E - S1
- E – S2
ordered
random
• Substrates bind to the enzyme at the same time to produce a
ternary complex
Ternary
complex
1. Ternary complex mechanism
Ternary complex mechanism
Ternary
complex
+ + +
Intermediate
transaminase
Ping- pong mechanism
1. Physico-chemical factors
➢ Substrate concentration
➢ Temperature
➢ pH
➢ Ionic strength
➢ Redox potential
TEMPERATURE
• Disruption of hydrogen bonds
• Disruption of the shape of the enzyme
Denaturation:
enzyme stability curve
• Optimal temperature t of most enzymes – similar or little higher
than the t of cells in which they occur
Shrimp
(cold water)
Bacteria
(hot springs)Human
Temperature
1. Physico-chemical factors
➢ Substrate concentration
➢ Temperature
➢ pH
➢ Ionic strength
➢ Redox potential
pH
alters the state of ionization of
charged amino acids in enzyme
Enz- + SH+ EnzSH
Effect of pH
Deviation from optimal pH - protein unwinding
- dissociation to subunits
- conversion to more compact form
LOSS of
activitySH+ + OH- S + H2O .......... high pH
Enz- + H+ EnzH ............... low pH
1. Physico-chemical factors
➢ Substrate concentration
➢ Temperature
➢ pH
➢ Ionic strength
➢ Redox potential
Ionic strength
• Concentration of salts influences enzyme activity because the salts affect
the hydration of proteins and consequently their solubility and shape of
molecules.
• Solubility of proteins at low ionic strengths increases with the
concentration of salt (so-called salting in). Increasing salt concentration
increases the solubility.
• At very high ionic strengths charges of protein molecules are shaded,
leading to the existence of very weak electrostatic interactions between
protein molecules, and thus solubility is reduced (salting out) .
1. Physico-chemical factors
➢ Substrate concentration
➢ Temperature
➢ pH
➢ Ionic strength
➢ Redox potential
REDOX POTENTIAL
Redox potential affects:
• some oxidizable groups especially –SH
• spacial arrangement of the whole enzyme molecule
• substrate binding (formation of –S-S- bonds)
Redox potential (RP) – a measure of the tendency of a chemical to
acquire electrons and thereby be reduced
The more positive the potential, the greater the species' affinity for
electrons and tendency to be reduced
Regulation of enzyme activity
A) Without the change in the quantity of enzyme
molecules 1) Physico-chemical factors
2) Presence of inhibitors and activators
3) Allosteric regulation of enzyme activity
4) Regulation by modification of enzyme molecule
5) Compartmentalization of enzymes
B) With the change of the number of enzyme
molecules1) Induction and repression
2) Regulated degradation of proteins
ENZYME INHIBITION
Nonspecific
Denaturation
Acids and bases
Temperature
Alcohol
Heavy metals
Reducing agents
Specific
Competitive
Noncompetitive
Uncompetitive
ReversibleIrreversible
Specific
DPFP, IAA
Irreversible inhibitors
▪ bind at the active site, or at a different site
▪ cannot be removed by dialysis
▪ often contain reactive functional groups forming covalent
adducts with AA side chains
▪ inhibition cannot be reversed
Examples of irreversible inhibition
❑ DIPFP (Diisopropyl fluorophosphate)- inhibits enzymes with
serine (acetyl cholinesterase) in the active site
❑ IAA (Iodoacetamide)- inhibits enzymes with cysteine in the
active site
❑ ASPIRIN - suppresses the production of prostaglandins and
thromboxanes due to its irreversible inactivation of the
cyclooxygenase
Irreversible inhibition - DIPFF
Diisopropyl fluorophosphate – binds to –OH group of
serine in the active site of enzyme
Diisopropyl fluorophosphate
• neurotoxin
• inhibitor of acetylcholinesterase (prolonged muscle
contraction - death)
Acetylcholine esterase
If the enzyme is inhibited, acetylcholine accumulates and nerve impulses cannot be
stopped, causing prolonged muscle contraction - paralysis occurs and death may
result since the respiratory muscles are affected.
NH2
NH2
Iodoacetamide
Irreversible inhibitions
Iodoacetamide – reacts with –SH groups in the active site
• proteins cannot form disulfide bonds
• toxic, carcinogen, reproductive damage
I
ARACHIDONIC ACID
Cyclooxygenase
ASPIRIN (Acetylsalicylic acid)
Inflammation,
Temperature
Irreversible inhibition - ASPIRIN
PROSTAGLANDINS
Active cyclooxygenase
Salicylic acid
Inactive cyclooxygenase
(Aspirin)
OH O- CO – CH3
Acetylation of the enzyme results in a steric block, preventing
arachidonic acid from binding
ENZYME INHIBITION
Nonspecific
Denaturation
Acids and bases
Temperature
Alcohol
Heavy metals
Reducing agents
Specific
Competitive
Noncompetitive
Uncompetitive
ReversibleIrreversible
Specific
DIPFP, IAA
Reversible
1) COMPETITIVE INHIBITION
• Inhibitor is structurally similar to the substrate
• The inhibitor competes with the substrate for the enzyme
active site
• Increasing concentration of substrate will outcompete the
inhibitor for binding to the enzyme active site
• Reversible inhibition
▪ Competitive Inhibitors work by preventing the formation of Enzyme-Substrate
Complexes because they have a similar shape to the substrate molecule.
KmI
1/2vmax
vmax = vImax Km < KI
m
Km
1/vmax
1/[S]
1/vI
-1
Km
-1
KmI
Lineweaver – Burk plot
Competitive inhibition
COO¯ COO¯
CH2 - 2H CH
+ FAD + FADH2
CH2 SDH CH
COO¯ COO¯
Succinate Fumarate
COO¯ COO¯
CH2 CO
COO¯ CH2
COO¯
Malonate Oxalacetate
COMPETITIVE INHIBITION
Competitive Inhibitors as Medicines
XANTHINE URIC ACID
Xanthine oxidase
ALLOPURINOLGOUT
CH3-CH2-OH CH3-C H CH3-C
ethanol acetaldehyde acetate
CH3-OH H-C H H-C
methanol formaldehyde formiate
CH2-OH CHO COOH
CH2-OH CH2-OH COOH
ethylene glycol glycol aldehyde oxalic acid
Ethanol – antidotum in methanol and
ethylene glycol poisoning
O O
O-
Alcohol dehydrogenase
OO
O-
Alcohol dehydrogenase
Alcohol dehydrogenase
Noncompetitive inhibition
Substrate
EnzymeInhibitor
site
Active
site
Enzyme binds substrate Enzyme releases products
Inhibitor
Inhibitor binds and
alters enzyme´s shape
Binding of substrate is
reduced
Inhibition:
Reaction:
• Inhibitor binds to the enzyme at a different place then
the substrate
• Inhibitor – structurally different from the substrate
No inhibitor
With inhibitor
Km = Kmv
max> v
max
II
1/v
1/[S]01/Km
1/V
I1
Noncompetitive inhibition
1/V
No inhibitor
• Noncompetitive inhibitors do not influence binding of S into the
active site of enzyme but they reduce the rate of its conversion to a
product. Therefore Km is unchanged and vmax is reduced.
• Because EIS decomposes more slowly than ES, the rate of
enzymatic reaction slows down
• Inhibitor binds only to the complex enzyme – substrate.
E + S [ES] [ES]I
I S
KmI < Km vI
max < vmax
Uncompetitive inhibition
Figure 4 – Illustrations
Uncompetitive inhibition
Uncompetitive inhibitors:
• Anticancer drugs
• Lithium
• vImax < v Km
I < Km
UNCOMPETITIVE INHIBITION
• multiple substrate mechanisms (ping-pong mechanism)
Normal
With inhibitor
With inhibitor Normal
Both the effective Vmax and effective Km are reduced with an inhibitor
V
1/V
Km Km -1/Km -1/Km
Regulation of enzyme activity
A) Without the change in the quantity of enzyme
molecules
1) Physico-chemical factors
2) Presence of inhibitors and activators
3) Allosteric regulation of enzyme activity
4) Regulation by modification of enzyme molecule
5) Compartmentalization of enzymes
B) With the change of the number of enzyme
molecules
1) Induction and repression
2) Regulated degradation of proteins
Allosteric enzymes
• Allosteric enzymes – change their conformation upon
binding of an effector (activator, inhibitor)
• Binding of the inhibitor to a site other than the active site changes
the shape of the active site – substrate cannot bind there
The allosteric inhibition
The allosteric activation
• Binding of the activator to a site other than the active site changes
the shape of the active site – substrate can bind there
Sigmoidal curve
Allosteric enzymes
Allosteric enzyme
Single subunit enzymes
◼ do not obey Michaelis-Menten kinetics
Allosteric enzymes
◼ display sigmoidal plots of the reaction velocity (v) versus
substrate concentration [S]
◼ the binding of substrate to one active site can affect the properties
of other active sites in the same molecule
◼ their activity may be altered by regulatory molecules that are
reversibly bound to specific sites other than the catalytic sites
Allosteric effectors of isocitrate
dehydrogenase
HH
Respiratory chain ATP
HO-C-COO-
CO2
Allosteric effectors of ICDH
ISOCITRATE
(+)NAD+ NADH + H+(-)
(+)ADP ATP(-)
(+)CITRATE
KREBS CYCLE
α-KETOGLUTARATE
ALLOSTERIC REGULATION
A B C D E P
E1 E2 E3 E4 E5
Feed-back regulationFeed-back inhibition
Feed-forward activation
• Metabolite B produced at the beginning of the metabolic pathway
can activate a downstream enzyme e.g.E4
Mechanism of activation of
allosteric enzymes
Cooperative model
(Concerted model)
Sequential model
• Both models postulate that enzyme subunits exist in one of
two conformations, tensed (T) or relaxed (R)
• Relaxed subunits bind substrate more readily than those in
the tense state.
S1 S2,S3
S4
Cooperative (concerted)model
(MONOD 1965)
T (Tensed) R (Relaxed)
S1 S2,S3
S4
Nonactive form Active form
❖ after binding a substrate a conformational change in one subunit is
necessarily conferred to all other subunits.
❖ all subunits must exist in the same conformation
SEQUENTIAL MODEL
(KOSHLAND 1966)
k1
k2
S1 + + S2
Sk4
k3
S
S+ S3
S
S
S
SS
SS
S4+k8
k7
T-conformation
nonactive
R –conformation - active
SEQUENTIAL MODEL
❖ substrate-binding at one subunit only slightly alters the structure of
other subunits so that their binding sites are more receptive to
substrate
❖ subunits need not exist in the same conformation
❖ conformational changes are not propagated to all subunits
❖ Substrate binding may result in an increased or a reduced affinity for
the ligand at the next binding site
Regulation of enzyme activity
A) Without the change in the quantity of enzyme
molecules
1) Physico-chemical factors
2) Presence of inhibitors and activators
3) Allosteric regulation of enzyme activity
4) Regulation by modification of enzyme molecule
5) Compartmentalization of enzymes
B) With the change of the number of enzyme
molecules
1) Induction and repression
2) Regulated degradation of proteins
4) Regulation by modification of enzyme
molecule
a) Limited proteolysis
b) Covalent modifications
a) Limited proteolysis
Inactive form of enzyme PROENZYME (ZYMOGEN) is
cleaved by proteases to the active enzyme
PROENZYME ACTIVE ENZYMEtrypsinogen trypsin (- pentapeptide)
pepsinogen pepsin (-1/5 molecule)
Enzymes produced by cells in the active form could damage own
protein structures (digestive enzymes)
Nonactive Active
substratesubstrate
Hydrolytic enzymes
PEPSIN
Pepsinogen Pepsin (peptide)
H+ (44 Aminoacids)
ENTEROPEPTIDASE
Trypsinogen Trypsin (6 AA)
TRYPSIN
Chymotrypsinogen Chymotrypsin + dipeptide
Similar mechanisms:
Proinsulin insulin pro-thrombin thrombin
Fibrinogen fibrin
4) Regulation by modification of enzyme
molecule
a) Limited proteolysis
b) Covalent modifications
b) Covalent modification of enzyme molecule
• Covalent attachment of a modifying group to a specific functional
group on the enzyme
A/ PHOSPHORYLATION, DEPHOSPHORYLATION
reversible modification, binding of a phosphate group to a
molecule by a specific kinase (in mammals)
B/ ADENYLATION – reversible binding of a nucleotide (e.g. AMP)
(in bacteria)
C/ ADP-RIBOZYLATION - reversible binding of ADP-ribosyl.
Donor of the ADP-ribosyl group is the coenzyme NAD+;
Phosphorylation,
Dephosphorylation
• Kinases - phosphorylate proteins
• Phosphatases - dephosphorylate
Phosphorylation
• on serine, threonine, tyrosine,
• conformational change of the structure
• on nonpolar part of proteins – increase
of polarity – change of conformation
Advantages of
phosphorylation/dephosphorylation:
◼ It is rapid (takes a few seconds)
◼ It does not require new proteins to be made or
degraded
◼ It is easily reversible
Regulation of enzyme activity
A) Without the change in the quantity of enzyme
molecules
1) Physico-chemical factors
2) Presence of inhibitors and activators
3) Allosteric regulation of enzyme activity
4) Regulation by modification of enzyme molecule
5) Compartmentalization of enzymes
B) With the change of the number of enzyme
molecules
1) Induction and repression
2) Regulated degradation of proteins
Compartmentalization of enzymes
◼ Enzymes are often compartmentalized - stored in a particular
organelle - they can find their substrates readily, don't damage
the cell, and have the right microenvironment to work well
◼ digestive enzymes of the lysosome work best at a pH around 5
which is found in the acidic interior of the lysosome (but not in
the cytosol, which has a pH of about 7.27).
Regulation of enzyme activity
A) Without the change in the quantity of enzyme
molecules
1) Physico-chemical factors
2) Presence of inhibitors and activators
3) Allosteric regulation of enzyme activity
4) Regulation by modification of enzyme molecule
5) Compartmentalization of enzymes
B) With the change of the number of enzyme
molecules
1) Induction and repression
2) Regulated degradation of proteins
Regulation of enzyme activity by changing
the number of enzyme molecules
1) Induction of enzyme synthesis
Constitutive enzymes – present at constant
concentrations (Krebs cycle)
Inducible enzymes – de novo synthesis of the enzyme
according to the need of a cell
2) Repression of enzyme synthesis – inhibition of
gene expression (actinomycins –inhibit transcription
streptomycin – inhibit translation)
- lactose
lactase
lactase
lactose
- lactose
lactase
lactase
lactose
Regulation of enzyme activity
A) Without the change in the quantity of enzyme
molecules
1) Physico-chemical factors
2) Presence of inhibitors and activators
3) Allosteric regulation of enzyme activity
4) Regulation by modification of enzyme molecule
5) Compartmentalization of enzymes
B) With the change of the number of enzyme
molecules
1) Induction and repression
2) Regulated degradation of proteins
Degradation of proteins in
eukaryotic cells
a) lysosomes - degradation of intracellular proteins
with a long half-life, extracellular proteins
associated with cell membrane
b) proteasomes – degradation of intracellular
proteins with a short half-life
Lysosomes
PROTEASOME
• Protein complex with proteolytic activity
• Located in the nucleus and the cytoplasm
• Proteins degraded in proteasome: transcription
factors, cyclins, proteins encoded by viruses...
Function:
Degradation of unneeded or damaged proteins by
proteolysis
19S regulatory subunit
19S regulatory subunit
20S catalytic subunit
Ubiquitin detachment
and protein unfolding
Regulation of enzyme activity by degradation
◼ Regulated by proteases – hydrolysis of peptide bonds
Proteins Peptides shorter peptides, aminoacids
proteases peptidases
endopeptidases – cleave intramolecular peptide bonds
Peptidases (trypsin, pepsin)
exopeptidases – cleave off a terminal amino acid
(carboxypeptidase A)
SPECIFICITY OF PROTEASES
• Ability to cleave peptide bonds next to a specific amino acid
Chymotrypsin – active site – hydrophobic
- preferentially cleaves peptide bonds next to aromatic
amino acids
Trypsin –in active center – negative charge
- cleaves peptide bonds from amino acids with positively
charged side chain
Chymotrypsin
Trypsin
Chymotrypsin
Trypsin
Chymotrypsin
Trypsin
1) INTRACELLULAR ENZYMES
• Stay in a cell in which they were synthesized
• Many occur only in some organs or cell organels
• In healthy organism – minimal concentrations in blood
ENZYMES
2) EXTRACELLULAR ENZYMES
• Secreted from cells of their origin
(e.g. in animals into digestive juice, blood...)
Enzyme Name Increased levels in disease
ALT
Alanine
aminotransferase Hepatopathy
AST
Aspartate
aminotransferase Myocardial infarction
LD
Lactate
dehydrogenase Myocardial infarction - LD1,2, hepatopathy - LD4,5
CK Creatine kinase
Myocardial infarction - CK-MB, skeletal muscle diseases -
CK- MM
ALP
Alkaline
phosphatase Diseases of the bile duct and liver, bone diseases
ACP Acid phosphatase Prostate tumors
AMS Amyláza Akútna pankreatitídaTissue specific enzymes
SOME ENZYME DEFECT DISORDERS
◼ Lactose intolerance – insufficient levels of lactase enzyme, which
breaks down the milk sugar - lactose
Symptoms of lactose intolerance
◼ stomach cramps,
◼ bloating,
◼ nausea,
◼ diarrhoea after consumption of milk products
Treatment
• lactose-free diet,
• pills with lactase enzyme
Sucrose (saccharose) intolerance
Sucrose intolerance – sucrase enzyme needed for proper
metabolism of saccharose (sucrose) and is not produced or the
enzyme produced is either partially functional or non-functional in
the small intestine.
Symptoms:
• chronic, watery, acidic diarrhea;
• gas;
• bloating
• abdominal pain.
Small
intestine
Large
intestine
Sucrose digestion
THERAPEUTIC ENZYMES
Many enzymes are produced on a large scale in microorganisms
like E. coli, or purified from other sources to treat diseases and
enzyme deficiencies.
Streptokinase and urokinase - dissolve dangerous blood clots in
people suffering from strokes and heart attacks.
Lysozyme - is used as an antibacterial agent as it specifically
dissolves bacterial cell walls
Chitinase is an antifungal which dissolves chitin in the cell walls
of fungi.