Download Enzymes

Enzymes
Josef Fontana
EC - 40
Overview of the seminar
• Introduction to the topic: Enzymes
–
–
–
–
Function of enzymes
Enzyme structure
Cofactors of enzyme groups
Izoenzymes and multienzyme complexes
• Nomenclature of enzymes
• Regulation on the cell level
– 1) Compartmentalization of metabolic pathways
– 2) Change of enzyme concentration (on the level of synthesis
of new enzyme)
– 3) Change of enzyme activity (an existing enzyme is activated
or inactivated)
– A) In relation to an enzyme kinetics
– B) Activation or inactivation of the enzyme
• Enzymes in a medicine
Introduction to the topic:
Enzymes
Function of enzymes
Enzyme is a biocatalyst:
the
reaction
The figure is found at: http://fig.cox.miami.edu/~cmallery/255/255enz/enzymology.htm (December 2006)
Function of enzymes
• They are special proteins produced
by living cells.
• They are catalysts:
– Increase the rate of chemical
reactions.
– Decrease the activation energy of
the reaction (EA).
– Reduce the time to reach the
reaction equilibrium.
– Are not consumed or changed by
the reaction.
• Help the reaction proceed under a
body T, p and pH.
• The action of most enzymes is very •
specific – substrate and reaction
specifity.
•
• They can be regulated.
Don´t change the ∆G of the
reaction.
Don´t change the equilibrium
position of the reaction.
The figure is found at: http://fig.cox.miami.edu/~cmallery/255/255enz/enzymology.htm (December 2006)
The figure is adopted from the book: Devlin, T. M. (editor): Textbook of Biochemistry with
Clinical Correlations, 4th ed. Wiley‑Liss, Inc., New York, 1997. ISBN 0‑471‑15451‑2
Enzyme catalysis
• Enzymes E are able to specifically
bind the reactants (their substrates
S) at the active site → complex E-S
(transition state, ↓ activation
energy) → destruction of complex ES to products P and E
The figure is found at:
http://stallion.abac.peachnet.edu/sm/kmccrae/BIOL2050/Ch1-13/JpegArt1-13/05jpeg/05_jpeg_HTML/index.htm (December 2006)
Introduction to the topic:
Enzymes
Enzyme structure
Structure and properties
• most of enzymes are proteins
The figure is found at: http://fig.cox.miami.edu/~cmallery/255/255enz/enzymology.htm (December 2006)
The figure is found at: http://fig.cox.miami.edu/~cmallery/255/255enz/enzymology.htm (December 2006)
Enzyme structure
• Enzymes are mostly proteins (exception: ribozyme catalytic active RNA).
• Some enzymes in addition to protein component
contain also non-protein component. According to
this we can divide enzymes into:
– Simple enzymes contain only protein (pepsin, trypsin,
ribonuclease).
– Complex enzymes contain protein and non-protein
component = cofactor.
• Cofactor is the non-protein part of the enzyme
molecule. It is necessary for its catalytic function.
Our body can not synthesize them often - therefore,
we eat their precursors – e.g. vitamins.
Cofactor can be
• 1) metal ion: Zn2+, Mn2+, Mg2+, Fe2+, Cu2+ (trace
elements)
• 2) organic molecule:
– coenzymes are slightly bound to the enzyme, undergo a
chemical change and are released from the enzyme
molecule, they are derivates of vitamins very often:
NAD(P)+, FAD, coenzyme Q, ...
– prosthetic groups are tightly bound to the enzyme and
remain associated with enzyme during the whole
reaction: heme, FAD,…
• Coenzyme + apoenzyme (inactive protein) →
holoenzyme (active enzyme)
The figure is found at:
http://stallion.abac.peachnet.edu/sm/kmccrae/BIOL2050/Ch1-13/JpegArt1-13/05jpeg/05_jpeg_HTML/index.htm (December 2006)
Introduction to the topic:
Enzymes
Cofactors of enzyme groups
Cofactors help to catalyze many
reactions
• Cofactors of oxidoreductases: NAD(P)+,
FAD, cytochromes (contain heme), Fe-S
complexes
• Coenzymes carrying C1 radicals:
tetrahydrofolate, vitamin B12, Sadenosylmethionine, biotin (cofactor of
carboxylases)
• Cofactors carrying acyl: lipoic acid (PDH
prosthetic group, α-KGDH) HSCoA,
pyridoxal phosphate (transaminases)
Cofactors of oxidoreductases
NAD+
NADP+
nicotinamide adenine dinucleotide
nicotinamide aden. dinucl. phosphate
(precursor: niacin = nicotinic acid)
H+
FAD
FMN
flavin adenine dinucleotide
flavin mononucleotide
2 H+
(precurzor: riboflavin = vitamin B2)
heme
Fe3+ + e- ↔ Fe2+
⇒
e-
Cofactors of transferases
ATP
GTP
TDP
adenosine triphosphate
guanosine triphosphate
thiamine diphosphate
/ phosphate
/ phosphate
/ C-fragment
PALP
pyridoxal phosphate
/ -NH2
(prekurzor: thiamine = vitamin B1)
(prekurzor: pyridoxine = vitamin B6)
THF
tetrahydrofolate
(prekurzor: folic acid)
/ C1-fragment
CoA
coenzyme A (HS-Co-A) / acyl
PAPS phosphoadenosine phosphosulfate / sulfate
Cofactors of
Lyases:
PALP pyridoxal phosphate (decarboxylases)
Ligases:
ATP adenosine triphosphate
→ acyl-CoA-synthetases
→ aminoacyl-tRNA-synthetases
biotin
= vitamin H (carboxylases)
Coenzymes and prosthetic group
NAD+ ↔ NADH + H+
nicotinamide adenine dinucleotide
coenzyme
FAD ↔ FADH2
flavin adenine dinucleotide
(vit. B2 = riboflavin)
prosthetic gr.
Other examples: coenzyme A, coenzyme Q, tetrahydrofolate, thiamine
diphosphate (vit. B1 = thiamine)
http://web.indstate.edu/thcme/mwking/vitamins.html
Prosthetic groups
Biotin (vit. H)
Heme
• Another example: pyridoxal phosphate (derivate
of vitamin B6)
http://web.indstate.edu/thcme/mwking/vitamins.html
Introduction to the topic:
Enzymes
Izoenzymes and multienzyme
complexes
Isoenzymes
• Some enzymes have variants called isoenzymes.
• They catalyze the same chemical reaction, but differ in
their primary structure and physico-chemical properties.
• Isoenzymes are:
– Produced by different genes (= true isozymes) or
– produced by different posttranslational modification (=
isoforms).
– Found in different compartments of a cell.
– Found in different tissues of an organism.
• For example lactate dehydrogenase (LD) has 5 isoenzymes:
LD1 – LD5.
• Isoenzymes are present in skeletal muscle, liver, heart,
kidney, erytrocytes. Isoenzymes can be separated by
electrophoresis.
– Can be oligomers of various subunits (monomers).
5 isozymes (various monomer
ratio)
The figure is found at: http://wine1.sb.fsu.edu/bch4053/Lecture26/isozymes.jpg (December 2006)
Separate enzymes of a mtb pathway
Multienzyme
complexes
This is Figure 17.6 from Garrett, R.H.; Grisham, C.M. Biochemistry; Saunders: Orlando,1995; page
553, found at http://www.uwsp.edu/chemistry/tzamis/enzyme_complex.html (December 2006)
example: 2-oxoacid dehydrogenase multienzyme complex
The figure is found at: http://faculty.uca.edu/~johnc/pdhrxns.gif (December 2006)
Nomenclature of enzymes
Nomenclature of enzymes
• 1) The first discovered enzymes were
named according to their source: Name
of enzyme + suffix -in
– Pepsin is found in the gastric juice (Greek
pepsis = digestion).
• 2) Enzymes were named according to
their substrate: Name of substrate +
specific suffix of enzymes –ase
– Lipase catalyzes the hydrolysis of lipids.
– Urease catalyzes the hydrolysis of urea.
Nomenclature of enzymes
• 3) In 1961 International Union of
Biochemistry recommended that
enzymes be systematically classified
according to the general type of
reaction they catalyze → 6 major
classes.
• Abbreviations of enzymes e.g. LD, ALT,
ALP
EC nomenclature
•
•
•
•
•
•
•
•
Each enzyme has a EC number (four-digit number)
We have 6 main classes:
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)
• Classification by a reaction catalyzed by the
enzyme
• E.g. Lactate dehydrogenase has the EC number
1.1.1.27
Systematic names
• Are made according to a special rules, they
specify a reaction catalyzed by the enzyme.
ATP : D-glucose phosphotransferase (EC 2.7.1.2)
→ transfers (2) phosphate (7) to an alcohol
group (1)
ATP + D-Glc → ADP + D-Glc-6-phosphate
(Glc-6-P)
Common name - glucokinase
EC nomenclature
• Oxidoreductases catalyze redox
reactions:
•
•
•
•
•
•
•
•
dehydrogenase (H- or H)
reductase
oxidase
peroxidase (various peroxides)
oxygenase (O2)
hydroxylase (= monoxygenase; -OH)
desaturase (-CH2CH2- → -CH=CH-)
alcohol dehydrogenase, catalase
EC nomenclature
• Transferases catalyze the transfer
of functional groups between donors
and acceptors
•
•
•
•
grouptransferase (e.g. aminotransferase)
kinase (= phosphotransferase)
Phosphorylase
Transketolase, transaldolase
EC nomenclature
• Hydrolases catalyze the hydrolytic
cleavage of substrates:
• Esterase
(R1-CO-O-R2)
• Phosphatase
(phosphate-O-R) → Pi !!!
• phosphodiesterase (R1-O-phosphate-O-R2)
• nuclease, peptidase, protease, glycosidase,
lipase, α-amylase
EC nomenclature
• Lyases (synthases) catalyze nonhydrolytic and non-oxidation cleavage or
synthesis of molecules (removing/addition
of the small molecule from/to substrate)
– decarboxylase (→ CO2)
– dehydratase (→ H2O)
– hydratase (-CH=CH- + H2O →
-CH(OH)CH2-)
– synthase
EC nomenclature
• Isomerases catalyze intramolecular
changes in substrate molecules
– epimerase (monosacharide → its epimer)
– mutase (rearangement of a phosphate
group)
EC nomenclature
• Ligases (synthetases) catalyze
synthetic reactions where 2
molecules are joined at 1 molecule,
synthesis requires an energy (ATP)
• A + B + ATP → A-B + ADP + Pi
– Polymerases
– synthetase
– carboxylase
Regulation on the cell level
Regulation on the cell level
•
1) Compartmentalization of metabolic
pathways
•
2) Change of enzyme concentration (on
the level of synthesis of new enzyme)
•
3) Change of enzyme activity (an
existing enzyme is activated or
inactivated)
Regulation on the cell level
1) Compartmentalization of
metabolic pathways
Compartmentalization of
metabolic patways
• Transport processes between compartments
• Various enzyme distribution
• Various distribution of substrates and
products (∼ transport)
• Transport of coenzymes
• Subsequent processes are close to each other
Regulation on the cell level
2) Change of enzyme
concentration (on the level of
synthesis of new enzyme)
Synthesis of new enzyme
molecule
• Enzyme concentration is much lower than the
concentration of substrate. The rate of an enzymecatalyzed reaction is directly dependent upon the
enzyme concentration.
• Induction by substrate or repression by product (on the
level of transcription)
– xenobiotics → induction of cyt P450
– heme → repression of delta-aminolevulate synthase
• Change in the rate of synthesis or degradation of the
enzyme - hormonal and nutritional factors
– Well-fed state: the liver improves its capacity to
synthesize fat.
– Fasting: ↓ in quantity of lipogenetic enzymes; enzymes of
gluconeogenesis are induced (↑synthesis).
Regulation on the cell level
3) Change of enzyme activity
(an existing enzyme is
activated or inactivated)
A) In relation to an enzyme kinetics
Enzyme kinetics
The figure is found at: http://fig.cox.miami.edu/~cmallery/255/255enz/gk3x15.gif (December 2006)
Enzyme kinetics - the curve can be
described by the equation:
• Michaelis constant KM corresponds to the substrate
concentration [S] at which velocity V is half of the maximum
velocity Vmax (when v = ½ Vmax). An enzyme with a high affinity
for its substrate has a low KM value.
• KM = mol/L
KM describes an affinity of the enzyme
to its substrate
! indirect proportionality !
The figure is found at: http://fig.cox.miami.edu/~cmallery/255/255enz/gk3x15.gif (December 2006)
Change of activity of an existing
enzyme
• A) In relation to an enzyme kinetics:
–
–
–
–
–
concentration of substrates (< Km)
pH and temperature changes
availability of coenzymes
consumption of products
substrate specificity - different Km
Substrate supply
• The rate of an enzymatic reaction increases as
the substrate concentration increases until a
limiting rate is reached.
Substrate supply
• The rate of an enzymatic reaction increases as
the substrate concentration increases until a
limiting rate is reached.
• Major determinant of the rate at which every
metabolic processes of the body operates:
–
–
–
–
blood fatty acids concentration → ketogenesis in
the liver
excessive amounts of substrates → synthesis of
excess fat
gluconeogenic substrates → rate of
gluconeogenesis
↑ Gln → ↑citrulline → ↑urea synthesis
Each enzyme has
temperature optimum
pH optimum
affinity to its substrate
The figure is found at:
http://stallion.abac.peachnet.edu/sm/kmccrae/BIOL2050/Ch1-13/JpegArt1-13/05jpeg/05_jpeg_HTML/index.htm
(December 2006)
Factors that influence
enzyme activity
• Temperature
– Most enzymes of warm-blooded animals have temperatures optimum of
about 37 oC. Protein structure of enzymes is denatured by heat (above 55
o
C)
• Hydrogen ion concentration (pH)
– Extreme values of pH (low/high) cause denaturation of protein. Optimum pH
of enzyme is a narrow pH range.
– Optimal pH for pepsin is 2.0 in the stomach, and for trypsin is 8.0 in small
intestine.
The figure is found at: http://www.carleton.ca/biology/2200/schedule.html (December 2006)
http://users
.rcn.com/jki
mball.ma.ultr
anet/Biology
Pages/E/Enz
ymes.html
Regulation on the cell level
3) Change of enzyme activity
(an existing enzyme is
activated or inactivated)
B) Activation or inactivation of the enzyme
Change of activity of an existing
enzyme
• B) Activation or inactivation of the enzyme:
• Covalent modification of the enzymes
– cleavage of an precursore (proenzyme, zymogen)
– reversible phosphorylation and dephosphorylation
(interconversion of enzymes by protein kinase or
protein phosphatase respectively)
• Modulation of activity by modulators (ligands):
– feed back inhibition
– cross regulation
– feed forward activation
Some enzymes are produced as
precursors (= Proenzymes or Zymogens)
Zymogenes (proenzymes) are nonactive forms of enzymes. They are
activated by cleavage of peptide from their molecule.
Trypsinogen → trypsin + hexapeptide
The figure is found at: http://wine1.sb.fsu.edu/bch4053/Lecture26/zymogen.jpg (December 2006)
or must be activated to be active
(e.g. by phosphorylation):
The figure is found at: http://fig.cox.miami.edu/~cmallery/150/memb/c11x11enzyme-cascade.jpg (December 2006)
Phosphorylation / dephosphorylation
• Some enzymes are active in a
phosphorylated form, some are inactive.
• Phosphorylation
–
–
protein kinases
macroergic phosphate as a donor of the
phosphate (ATP !)
• Dephosphorylation
–
–
protein phosphatase
inorganic phosphate is the product !
Reversible covalent
modification:
A)
• phosphorylation by
a protein kinase
• dephosphorylation by
a protein phosphatase
B)
• phosphorylated enzyme
is either active or inactive
(different enzymes are
influenced differently)
The figure is found at:
http://stallion.abac.peachnet.edu/sm/kmccrae/BIOL2050/Ch1-13/JpegArt1-13/05jpeg/05_jpeg_HTML/index.htm (December 2006)
Modulators of enzyme activity
(activators, inhibitors)
• Isosteric modulation: competitive
inhibition
• Allosteric modulation:
–
change of Km or Vmax
–
T-form (less active) or R-form (more
active)
• Important modulators: ATP / ADP,
NAD+ / NADH + H+
Inhibitors
• Some chemical compounds can act as enzyme
inhibitors. Enzyme inhibition:
– a) irreversible
– b) reversible
• Irreversible inhibition
– Irreversible inhibitors react with enzyme and form a
covalent adduct with protein or metal ion.
– HCN inactives iron-containing enzymes because it
binds to Fe2+ in heme. HCN blocks cellular respiration
(cytochrome c oxidase).
– The nerve gases inhibit transmission in nerve system
because they block specific enzymes (tabun, sarin).
Inhibition of enzymes
The figure is found at:
http://stallion.abac.peachnet.edu/sm/kmccrae/BIOL2050/Ch1-13/JpegArt1-13/05jpeg/05_jpeg_HTML/ind
(December 2006)
Competitive inhibition
• Competitive inhibitor „competes“
with a substrate S for binding at
enzyme´s active site.
• It is bound to an active site but
not converted by the enzyme.
• Vmax value is unchanged.
• KM value is elevated (it is
necessary to add more S to reach
the original enzyme activity) ↓afinity of enzyme to its S.
• If concentration of a substrate is
increased the inhibition is
decreased.
The figure is found at: http://www.steve.gb.com/science/enzymes.html (December 2006)
Noncompetitive inhibition
• Inhibitor I binds at a site other
than the substrate-binding site.
• I binds with an equal affinity to
the free enzyme and to the ES
complex.
• Vmax is decreased (it is related to
decreasing of concentration of an
active enzyme).
• KM value is unchanged.
• Inhibition is not reversed by
increasing concentration of
substrate (no Km change).
The figure is found at: http://www.steve.gb.com/science/enzymes.html (December 2006)
Summary of the inhibition
The figure is found at: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html
(December 2006)
Allosteric enzyme:
a) monomeric, b) oligomeric
The figure is adopted from the book: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical
Correlations, 4th ed. Wiley‑Liss, Inc., New York, 1997. ISBN 0‑471‑15451‑2
Allosteric enzyme in T and R conformations:
modulators shift the equilibrium
inhibitors
have a
greater
affinity
for
T-state
activators
and
substrates
have a
greater
affinity for
R-state
The figure is adopted from the book: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th
ed. Wiley‑Liss, Inc., New York, 1997. ISBN 0‑471‑15451‑2
Allosteric
regulation
• activator is
„a positive
modulator“
• inhibitor is
„a negative
modulator“
! the curve of allosteric enzymes is sigmoidal not hyperbolic !
The figure is found at: http://www-biol.paisley.ac.uk/Kinetics/Chapter_5/chapter5_2_2.html (December 2006)
Allosteric effectors (negative or
positive)
• ↑glucose: inhibits glycogen phosphorylase, activates
glycogen synthase (= mtb of glycogen)
• ↑fructose-2,6-bisphosphate (↑ if insulin is ↑):
inhibits fru-1,6-bisphosphatase (=
gluconeogenesis), activates 6-PFK-1 (= glycolysis)
• ↑citrate: inhibits 6-PFK-1 (= glycolysis), activates
acetyl-CoA carboxylase (= fatty acid synthesis)
• ↑acetyl-CoA: inhibits pyruvate dehydrogenase,
activates pyruvate carboxylase (= activation of
gluconeogenesis)
• ↑malonyl-CoA inhibits carnitine palmitoyl transferase
I (= β-oxidation)
Inhibition as a regulation of
metabolic pathways
• Inhibition by products or intermediates:
– a) feedback regulation
– b) cross-regulation
– c) feedforward regulation
• Inhibition by
– d) reversible covalent modification
– e.g. phosphorylation/dephosphorylation
http://users.rc
n.com/jkimball.
ma.ultranet/Biol
ogyPages/E/Enz
ymes.html
Enzymes in a medicine
Enzymes in a medicine
• Determination of enzyme activity in
blood.
• Enzymatic analytical methods.
• Enzyme therapy.
Diagnostic applications of
enzymes
•
The measurement of enzyme activity in body fluids (plasma,
serum) has become an important tool in medical diagnosis. Under
normal conditions the concentrations of enzymes is low in blood.
An abnormally high level of a particular enzyme in the blood
often indicates specific tissue damage (hepatitis, myocardial
infarction,....). Some important enzymes for clinical diagnosis:
Enzyme assayed
Organ or tissue damaged
α-amylase (AMS)
pancreas
alkaline phosphatase (ALP)
bone, liver
creatine kinase (CK)
muscle, heart
lactate dehydrogenase (LD)
heart, liver
alanine aminotransferase (ALT)
liver
aspartate aminotransferase (AST)
heart, liver
Enzymatic activity
• Units
– 1 katal = 1 mole of a substrate
transformed per 1 sec
– 1 IU = 1 μmole of a substrate
transformed per 1 minute
• 1 katal = 1 mole / 1 sec
= 106 μmole /1 sec
•
= 60 x 106 μmole /1 min (= 60 sec)
•
• 1 katal = 6 x 107 IU
Diagnostic applications of
enzymes
• Normal (physiological) activity of
ALT in blood is up to 0.73
µkat/L.
• Activity of ALT in serum during
acute virus hepatitis is 50x
higher than normal activity!
Markers of cardiac damage
• Při poškození buňěk myokardu se původně
intracelulární látky dostávají do oběhu a my je
tak můžeme stanovit při odběru krve.
• Stanovují se následující látky:
• Kreatinkináza (CK) a její izoenzym CK-MB
• Troponin T nebo troponin I = výborné markery
poškození myokardu, jsou totiž odlišné od
troponinů v kosterní svalovině (>99% specifita
pro myokard)
• Dnes už jen historický význam mají AST a
myoglobin.
Summary
Regulation of enzyme activity
• availability of a substrate and its
concentration
• induction of synthesis of a regulatory
enzyme
• activation of enzyme precursors
• covalent modification of enzymes
• competitive inhibition
• allosteric regulation