BIOCATALYSTS · 1897 –Eduard Buchner –ability of yeast extracts that lacked any living yeast...
Transcript of BIOCATALYSTS · 1897 –Eduard Buchner –ability of yeast extracts that lacked any living yeast...
ENZYMES BIOCATALYSTS
2009/2010
INGRID ŽITŇANOVÁ
ENZYMOLOGY
HISTORY
17th -18th century – digestion of meat caused by stomach secretions
- conversion of starch to glucose by saliva
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
Louis Pasteur
Eduard Buchner
• 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:
1) More efficient - higher reaction rate
2) Milder reaction conditions (20-40C, 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?
• Glucose oxidation in solution and in human
organism
Example of enzymes effectiveness
• The oxidation of a fatty acid to carbon dioxide and
water
in the body: reaction takes place smoothly and rapidly within a
narrow range of pH and temperature
in the test tubes: extreme pH, high temperature and corrosive
chemicals
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
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
serve several apoenzymes:
NAD+ (nicotinamide adenine dinucleotide) - a coenzyme 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)
Biocytin CO2 fixation
Coenzyme A acyl group carrier
B1
B2
B3
pantothenic acid
B5
H
ACTIVE SITE
Active siteSubstrate
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
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
Binding sites (substrate, cofactor)
Catalytic site
ENZYME SUBSTRATE
ACTIVE SITE
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“
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 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)
b. Linkage specific – particular type of chemical bond regardless
. of the rest of the molecular structure (peptidase, esterase)
SPECIFICITY OF EFFECT (cofactor responsible)
Each enzyme can catalyze only a certain type of a
chemical reaction
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
2) Induced Fit Model (Daniel Koshland 1958)
This model assumes continuous changes in 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
• Creating an EC complex reduces bonds in the substrate and
makes the substrate easier to convert to the product – it lowers
the activation energy.
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)
ENZYME NOMENCLATURE
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
ENZYME COMPLEXES
Formation of a binary complex between 2 enzymes of a certain
reaction chain
Aldolase + glycerolphosphate dehydrogenases
Multienzyme complexes
pyruvate dehydrogenase system
complex of fatty acid synthesis (7 enzymes)
Multienzyme complex of pyruvate
dehydrogenase
MULTIENZYME COMPLEX
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
Enzyme concentration
Temperature
pH
Ionic strength
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 M-M 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]
• 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
Enzyme concentration
Temperature
pH
Ionic strength
E
No enzyme
enzyme
Concentration of Enzyme
• Substrate is present in a large excess
Co
nc.
of
pro
du
ct
1. Physico-chemical factors
Substrate concentration
Enzyme concentration
Temperature
pH
Ionic strength
Optimal
temperature
TEMPERATURE
• Disruption of hydrogen bonds
• Disruption of the shape of the enzyme
Denaturation:
enzyme stability curve
kinetic energy curve
• Optimal 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
Enzyme concentration
Temperature
pH
Ionic strength
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
Enzyme concentration
Temperature
pH
Ionic strength
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
DIPFP, 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
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
Diisopropylfluorophosphate
• neurotoxin
• inhibitor of acetylcholinesterase (prolonged muscle
contraction - death)
Acetylcholine esterase
NH2
NH2
Iodoacetamide
Irreversible inhibitions
Iodoacetamide
• 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
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 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 INHIBITION
Active center
ENZYME
Inhibitor
Complex Enzyme-Inhibitor
ENZYME
SubstrateS
S
I I
S
S
ENZYME ENZYMEI
IS
S
S S
S
S
S
S
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
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
(Hg2+, Zn2+, Cu2+,Pb2+, CN¯, CO)
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
Enzyme Inhibitions (Mechanisms)
I
I
S
S
S I
IS
E
Different siteCompete
for an active site
Inhibitor
Substrate
E + S→ES→E + P
+I
↓
EI
←
↑
E + S→ES→E + P
+ +I I
↓ ↓
EI+S→EIS
←
↑ ↑
E + S→ES→E + P
+I
↓
EIS
←
↑
E
I
S
Juang RH (2004) BCbasics
I Competitive NoncompetitiveI I Uncompetitive
E
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 conformational
ensemble upon binding of an effector
catalytic center – binds substrate
binds activator
allosteric center
binds inhibitor
subunits
Active siteallosteric
center
• 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
Activator - stabilizes R (relaxed)
Inhibitor stabilizes T (tense)
Sigmoidal curve
Allosteric enzymes
Allosteric enzyme
Isosteric enzyme
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 INHIBITION
A B C D E P
E1 E2 E3 E4 E5
Feed-back regulationFeed-back regulation
Feed-forward activation
• Metabolite B produced at the beginning of the metabolic pathway
can activate a downstream enzyme e.g.E4
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 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
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
Ligand 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
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
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
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
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...)