Enzymes
BASICS of enzymology Enzyme
Kinetics
Biochemistry-3
Two basic conditions of life
1. Living organisms must be capable of
self-replication
2. Organisms must be capable of
catalyzing chemical reactions efficiently
and selectively
Biochemistry-3
-Enzymes are biological catalysts systems enabling
chemical transformations. They also allow the
transformation of one form of energy to another.
-For enzymes characteristic catalytic power and specificity.
- The catalytic power of the enzyme is defined as the ratio of
the rate of reaction catalyzed by the enzyme reaction rate
and uncatalyzed.
- Catalysis takes place in the enzyme molecule called
active site.
- A substance which catalyses the conversion is called a
substrate.
- Nearly all known enzymes are proteins (RNA are probably
the earliest catalysts - ribozymes).
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4
General properties of enzymes
• proteins
• biocatalysts
specific (due to the substrate effect)
highly active
operate under mild conditions
may be regulated
in vitro - sensitive to external
conditions
Biochemistry-3
5
Enzymes are different
proteins
-simple proteins
- with covalently bound prostetic group
- metalloenzymes
- oligomeric, multienzyme complexes
- associated with membranes
- differently distributed in body/cell
- forms isoforms Biochemistry-3
6
Enzymes are highly efficient catalysts
• Enzymes are highly efficient catalysts
• reduce the activation energy  accelerate reactions
-efficiency by several orders of magnitude higher than that of other catalysts
- reaction with the enzyme is about 10 6-10 14 faster than without enzyme
do not affect the equilibrium constant K
- relatively low stable
If the reaction biol. systems were
catalyzed by enzymes, they would be
so slow that it could not ensure the
existence of living matter
Biochemistry-3
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Enzymes work under mild conditions
• atmospheric pressure
• narrow temperature range - about 37 ° C.
•
above 50 ° C usually denatured
•  narrow pH range
• pH optimum
Biochemistry-3
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There is a possibility of regulation
The enzyme activity
-activators
-inhibitors
- covalent
modification
(phosphorylation)
The amount of enzyme
-regulation of protein synthesis
- enzyme proteolysis
- some hormones - inducers ×
repressors
Biochemistry-3
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The specificity of enzymes is double
Consecutive, effects
of the possible
reactions
they catalyze only a
single substrate
Substrate specificity
-possible substrates
for a reaction can
choose one (or a
single group of
substrates)
-often stereospecific
Biochemistry-3
10
Enzymes are stereospecific catalysts
• There are two types of transformations:
conversion of an achiral substrate to a chiral product (= single
enantiomer), for example. pyruvate  L-lactate
- transformation of a chiral substrate (only one enantiomer) of
the product (also important for pharmacology)
L-alanin  pyruvate (D-alanin do not interact)
D-glucose   pyruvate (L-glucose do not interact)
chiral signal molecule  complex with receptor  biological answer
chiral drug(ant)agonista  complex with receptor  farmacological answer
Biochemistry-3
11
Example:
hydrogenation of pyruvate
in vitro
Non-enzymatic
created racemate
(D, L-lactate)
in vivo
enzyme
created only one
enantiomer (L-lactate)
Biochemistry-3
12
Hydrogenation of pyruvate in vitro
adition of hydrogen to planar substrate from both sides
H
C C
CH3
O
O
HO
H
C
COOH
CH3
HHO + C
COOH
CH3
OHH
L-laktát + D-laktát
There are statistically the same probability approach reagent from
both sides of a planar substrate - therefore arises racemate
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, Enzymy s. 3
L-lactate and D-lactate
Biochemistry-3
13
Hydrogenation of pyruvate in vivo
(anaerobic glycolysis) addition of hydrogen to the planar
substrate from one side only
H
C C
CH3
O
O
HO
C
COOH
CH3
HHO
enzym
L-laktát
vzniká jediný
stereoizomer
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 3
enzyme
L-lactate
Only 1
stereoisomereBiochemistry-3
14
Example: Non-enzymatic hydration of
fumarate
in vitro formed racemic D, L-malate
fumarate
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 3
C
C
COOHH
HOOC H
H
H
O
H
H
O
adice z pravé
strany
C
COOH
CH2COOH
OHHC
COOH
CH2COOH
HHO
adice z levé
strany
L-malate D-malate
Biochemistry-3
15
Enzyme hydration of fumarate (CC)
occurs in vivo, only one enantiomer (L-malate)
Reagent
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009,
Enzymy s. 3
C C COOH
H
H
HOOC
H
O
H
Enzyme
Substrate
Biochemistry-3
16
Binding of a chiral substrate (drug) into the active
site of the enzyme
active drug
Non-active drug
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 4
Active side of
enzyme
R R
x
active enantiomer nonactive enantiomer
R
x
chiral substrate
vazebná
místa enzymu
Biochemistry-3
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The nomenclature of enzymes
trivial
extension -in, ase
pepsin, trypsin
amylase, lipase
recommended trivial names
system
terminal -ase
name contains
information on:
1. substrate
2. type of response
Biochemistry-3
18
examples of names
• Recommended trivial name: alcohol dehydrogenase
System name: ethanol: NAD + -oxidoreductase
Reaction: ethanol + NAD +  acetaldehyde + NADH + H +
• Recommended trivial name: alanine aminotransferase (ALT)
System name: L-alanine-2-oxoglutarate aminotransferase
Reaction: L-alanine + 2-oxoglutarate  pyruvate + L-glutamate
Biochemistry-3
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Classification of enzymes:
Six classes according to the type of reaction
1. oxidoreductase
2. transferase
3. hydrolases
4. lyase
5. Isomerase
6. ligase
TEST, examples
Biochemistry-3
20
1. Oxidoreductases
• redox conversion of substrates
various mechanisms, subclasses:
• dehydrogenases, transmit two H atoms
(dehydrogenase)
• oxidase, transmitted electrons from substrate to
substrate (cytochrome c oxidase)
• oxygenase, incorporated into the substrate O atom
(monooxygenases, dioxygenase)
• Peroxidase (degradation of H2O2)
Biochemistry-3
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2. Transferases
• transfer groups from one substrate to another
aminotransferase, methyl, amino, glucose ...
kinase - phosphorylation substrates -PO3
2- transfer
from ATP to the OH group of the substrate
Biochemistry-3
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Example: Phosphorylation of Glu
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 5
O OH
OH
OH
OH
HO
O OH
OH
OH
OH
O
P
O
O
O
glukokinase
+ ATP + ADP
glucose glucose-6-P
Biochemistry-3
23
3. Hydrolases
• hydrolytically cleaves the links
established by condensation
• peptide, ester, glycosidic
protease, amylase, lipase, lysozyme
• phosphatase - cleaves phosphate esters
Biochemistry-3
24
Example: Phosphatase
glukosa-6-fosfatasa
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 5
O OH
OH
OH
OH
HO
O OH
OH
OH
OH
O
P
O
O
O
glucoseglucose-6-P
+ H2O OH
P
O
O
O
+
Glucose-6-
phosphatese
Biochemistry-3
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4. Lyases
• non-hydrolytic cleavage and formation
of C-C bonds, C-O, C-N
• cleaved from the substrate or to bring it a
small molecule (CO2, H2O)
• for example. fumaratehydratase
Biochemistry-3
26
Example: Hydratation of fumarate
on malate (fumaratehydratase)
+ H2O
COOH
C H
CH2
HO
COOH
C
C
COOHH
HOOC H
fumaráthydratasa
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 6
fumaratehydratase
Biochemistry-3
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5. Isomerases
• intramolecular rearrangements of atoms
glucose-4-epimerase (epimerization of
glucose)
UDP-glucose  UDP-galactose
Biochemistry-3
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6. Ligase
• formation of energy-intensive ties with
decomposition energy compounds (ATP)
•
pyruvate carboxylase
pyruvate + CO2 + ATP + H2O  oxalacetate + ADP + Pi
Biochemistry-3
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Three different components in the
enzyme reaction
enzym
substrát kofaktor  produkt kofaktormodif+ +
1. Substrate (s) - a low molecular weight
2. cofactor - low molecular
3. enzyme - high molecular weight, coordinates and accelerates the
reaction
Directly interact
Note .: some reactions proceed without cofactor (eg.
hydrolysis), the substrates can be a high molecular weight
substrate cofactor product Cofactor-modif
enzyme
Biochemistry-3
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Cofactors of enzymes
• low molecular weight non-protein compounds
- transmit e- or 2H --- oxidoreductase
- transfer of groups --- transferase
tightly bound - prosthetic group
loosely coupled - coenzymes (co-substrate)
TEST
Biochemistry-3
Coenzymes and prosthetic
groups
Biochemistry-3
Nomenclature
• Cofactor: nonprotein component of enzymes
• Cofactor - a co-catalyst required for enzyme activity
1) Coenzyme - a dissociable cofactor, usually organic
2) Prosthetic group - non-dissociable cofactor
• Vitamin - a required micro-nutrient (organism cannot
synthesize adequate quantities for normal health may
vary during life-cycle).
– water soluble - not stored, generally no problem with
overdose
– lipid soluble - stored, often toxic with overdose.
• Apoenzyme - enzyme lacking cofactor (inactive)
• Holoenzyme - enzyme with cofactors (active)
TEST
Biochemistry-3
Vitamins are precursors of cofactors
Biochemistry-3
34
Vitamins and cofactors of oxidodeductases
Vitamine Cofactors Function of cofactors
Niacin
Niacin
Riboflavin
-----
-----
-----
-----
-----
-----
-----
NAD+
NADPH + H+
FAD, FMN
tetrahydrobiopterin
molybdopterin
lipoate
ubichinon
hem cytochrom
nonhem Fe a S
2 GSH
acceptore 2H
donore 2H
accceptore 2H
donor 2H
e transport
acceptore 2H
2 electron transport (and 2H+)
1 electron transport
1 electron transport
donore 2H
Biochemistry-3
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Cofactors of oxidoreductases
always exist in two forms
oxidized  reduced
form a redox couple
Biochemistry-3
Nicotinic Acid/Nicotinamide Coenzymes
• These coenzymes are two-electron carriers
• They transfer hydride anion (H-) to and
from substrates
• Two important coenzymes in this class:
• Nicotinamide adenine dinucleotide (NAD+)
• Nicotinamide adenine dinucleotide
phosphate (NADP+)
Biochemistry-3
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Structure of NAD+
NOVÁK, Jan. Biochemie I.
Brno: Muni, 2009, Enzymy s. 8
O
OH O
N
CONH2
CH2 O P O P O
OH
O O
OH
CH2
H O
OH OH
N
N
N
N
NH2adenine
pyridinium
ribose
Diphosphor. acid
Adition of hydride anion
N-glycosidic bond
ester
anhydrid
ester
ribose
N-glycosidic bond
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NAD+ NADH (+H+)
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 8
N
CONH2
H
N
CONH2
HH
Oxidized form Redused form
H
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Dehydrogenation with NAD+
• substrate loses two H atoms from the groups:
primary alcohol group CH2-OH
secondary alcohol group> CH-OH
a secondary amino group> CH-NH2
there is a double bond
Biochemistry-3
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Dehydrogenation of ethanol
(alcoholdehydrogenase)
H3C C
H
H
O
H
+ NAD H3C C
H
O
+ NADH+H
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 8
Biochemistry-3
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Dehydrogenation of glutamate
(glutamatedehydrogenase)
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 9
C HN
H
H
COOH
CH2
CH2
COOH
+ NAD
CHN
COOH
CH2
CH2
COOH
+ NADH + H
2-amino acid 2-imino acid
Biochemistry-3
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NAD+-dependent enzyme
• Citrate cycle:
isocitratedehydrogenase
2-oxoglutaratedehydrogenase
malatedehydrogenase
• Glykolysis:
glyceraldehyd-3-P dehydrogenase
laktatedehydrogenase
• Detoxication of ethanol:
alcoholdehydrogenase
acetaldehyddehydrogenase
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NADPH + H+ hydrogenation agent
• donor 2H hydrogenation
reducing cofactor synthesis (FA, cholesterol)
regeneration of GSH in erythrocytes!
cofactor hydroxylation reactions:
cholesterol bile acids
kalciol -- calcitriol
xenobiotic -- hydroxylated xenobiotic
general scheme hydroxylation:
R-H + O2 + NADPH+H+  R-OH + H2O + NADP+
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FAD is dehydrogenation agent
• flavinadenindinucleotide
• cofactor dehydrogenase
• Dehydrogenation of -CH2-CH2- group
• 2H are bindint to 2N of riboflavine
Biochemistry-3
Prostetic group
45
Structure of FAD
N
N N
NH
H3C
H3C
O
O
CH2
CH OH
CH
CH
OH
OH
CH2 O P O
OH
O
P
OH
O
O
CH2
O
OH OH
N
N
N
N
NH2
Biochemistry-3
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FAD FADH2
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 10
N
N
N
NO
O
CH3
CH3
H
Oxidized form Reduced form
N
N
N
NO
O
CH3
CH3
H
H
H
1
10
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Dehydrogenation of succinate to fumarate
COOH
CH2
CH2
COOH
+ FAD
C
C
COOHH
HOOC H
+ FADH2
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 10
Biochemistry-3
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Tetrahydrobiopterine (BH4)
is hydrogenation agent
• coffactor of hydroxylation reactions
• gives 2H on O (water is produced)
• Oxidation on chinoid
dihydrobiopterine
Biochemistry-3
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Dehydrogenace
tetrahydrobiopterine
N
N
N
NN
O
CH3
OH
OH
H
H
H
H
H
- 2H
H
H
N
N
N
NHN
O
CH3
OH
OH
tetrahydrobiopterin
(BH 4)
dihydrobiopterin
(BH 2)
chinoid
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 10
Biochemistry-3
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Hydroxylation of
phenylalanine
COOH
CHH2N
CH2
H
+ O O + BH4
COOH
CHH2N
CH2
OH
+ +H2O BH2
phenylalanin tyrosin
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 11
Biochemistry-3
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Coenzym Q (ubichinon)
• a derivative of 1,4-benzoquinone
- component of the respiratory chain
- gradually accepts an electron and
proton (2x)
- reduces the semiubichinon and
ubiquinol
Biochemistry-3
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Hydrogenation of ubichinone
O
O
CH3
RH3CO
H3CO
OH
O
e H+e H+
OH
OH
ubichinon semiubichinon ubichinol
R = polyisoprenoid chain  lipophilic
elektrone (e-) and protone (H+) have different origine:
elektrone from red. coffactors (=nutrients), H+ from matrix
of mitochond.
no arom. ring arom. ring + radical diphenol
NOVÁK, Jan. Biochemie
I. Brno: Muni, 2009,
Enzymy s. 11
Biochemistry-3
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Hem of cytochromes
• transfers one electron
cytochromes are hemoproteins,
reversible transition occurs between Fe2 +
and Fe3 +
N N
NN
Fe 2+
N N
NN
Fe 3+
NOVÁK, Jan.
Biochemie I.
Brno: Muni,
2009, Enzymy s.
11
Biochemistry-3
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Non hem Fe – claster of Fe2S2
only one atome of Iron changes oxid. number
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 11
S
Fe
S
S
Fe
S
S
S
CysCys
Cys Cys
3+ 3+
Oxidized form Reduced form
S
Fe
S
S
Fe
S
S
S
CysCys
Cys Cys
3+ 2+
+ e
- e
Biochemistry-3
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Molybdopterine
• the pteridine system heterocycle
bonded with molybdenum
oxygenase cofactor
for example. xanthine oxidase,
sulfitoxidase
Biochemistry-3
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Xanthinoxidase: oxygenation of purine
N
N
N
N
OH
H
N
N
N
N
OH
HO
H
N
N
N
N
OH
HO OH
H
hypoxanthin  xanthin  uric acid.
Biochemistry-3
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Sulfitoxidase: formation of sulfate
anion
cysteine
HSO3
- + H2O  SO4
2- + 3 H+ + 2 eplazma
(0,5 mmol/l)
urine
ECT
Reduction of
Mo
Biochemistry-3
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Lipoate
• cyclic disulfide (S-S)
- 1,2-dithiolane-3-pentanoic acid
- amide linkage to lysine enzyme
- adoption 2H has two SH groups
- part of a complex oxidative
decarboxylation of 2-oxo acids
(pyruvate, 2-OG)
Biochemistry-3
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Hydrogenation of lipoate
S S
CONH Lys Enzym
2H
CONH Lys Enzym
SS
H H
lipoate
dihydrolipoate
Biochemistry-3
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Glutathione (GSH)
• tripeptide
• γ-glutamylcysteinylglycin
• cofactor of glutathionperoxidase
• Reduction of H2O2 to water
compounds with –SH reduction properties
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Dehydrogenation of 2 molecules of
GSH
HOOC
N
N COOH
O
H
CH2
S
O
H
NH2
N
NHOOC
O
H
CH2
S
O
H
NH2
COOH
HOOC
N
N COOH
O
H
CH2
SH
O
H
NH2
2 - 2H
Biochemistry-3
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Vitamine cofaktor group
---
---
folic acid
Biotin
Thiamin
Pyridoxin
Pantothen.acid
---
[Methionin]
cyanokobalamin
ATP
PAPS
H4-folate
carboxybiotin
thiamindiP
pyridoxalP
CoA-SH
dihydrolipoate
SAM
Methylcobalamin
-PO3
2-
-SO3
2C1
group
CO2
aldehyd
-NH2
acyl
acyl
-CH3
-CH3
Vitamins and cofactors of transferases
Biochemistry-3
63
Pyridoxalphosphate is cofactor of
transaminases
R CH
NH2
COOH
N
CH2O
H3C
HO
C
OH
P
OH
O
OH
aldimin (Schiff base)
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 13
Biochemistry-3
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Scheme of 2 phases of transamination
Schiff base
NOVÁK, Jan. Biochemie
I. Brno: Muni, 2009,
Metabolismus AK s. 10
AA
2-Oxo-acid
CH
NH2
COOHR R C COOH
CH2
N
R CH COOH
CH
N H2O
R C
O
COOH
CH2NH2
C
OH - H2O
pyridoxal pyridoxamin
C
OH
CH2NH2
HOOC C
O
CH2CH2COOH
- H2O
H2O
HOOC CH CH2CH2COOH
NH2
2-oxoglutarate glutamate
pyridoxamine pyridoxal
Biochemistry-3
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ATP (adenosintriphosphate)
• 2 importances :
• macroergic compound
• cofactor of kinases - phosphorylation
reagent
Biochemistry-3
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Structure of ATP
N
N
N
N
NH2
O
OHOH
OP
O
O
OP
O
O
O
P
O
O
O N-glycosidic
bond
ester
anhydrid
Biochemistry-3
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Phosphorylation of
substrate
http://www.piercenet.com/media/S
erine%20Phosphorylation.jpg
Biochemistry-3
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PAPS is the sulfation agent
N
N
N
N
NH2
O
OHO
CH2
P
O
O O
OPO
O
O
S
O
O
O
• 3'-5'-fosfoadenosin
phosphosulfate
- mixed anhydride of H2SO4
and H3PO4
- esterification of the
hydroxyl groups of the acid.
sulfuric
Biochemistry-3
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Carboxybiotin
• cofactor for carboxylation reactions
carboxylation of biotin requires ATP
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 14
N NH
S
O
COOH
C
O
HO
HN NH
S
O
COOH
CO2 ATP
ADP + P
biotin carboxybiotin
Biochemistry-3
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carboxybiotin is
cofactor of
carboxylation reactions
N NH
S
O
COOH
C
O
HO
+
NOVÁK, Jan.
Biochemie I. Brno:
Muni, 2009
Biotin COOH
H3C C
O
COOH
Biotin H
CH2 C
O
COOHHOOC
pyruvate oxalacetate
pyruvatcarboxylase
Biochemistry-3
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Distinguish
• Carboxylation
• carboxybiotin
• Decarboxylatione
enzymatic (AA - pyridoxalphosphate, 2-oxo acid. -
TDP)
• non enzymatic (spontanous, without enzyme and
cofactor, ex. acetoacetate → aceton)
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Coenzym A (CoA-SH)
• transfers the acyl
- bonded to the sulfur atom
thioester bond
- acyl-CoA is activated acyl
- eg. acetyl-CoA
R C
O
Biochemistry-3
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Coenzyme A
pantoová
kyselina
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 14
HO
P
O
O
OCH2C
HS CH2 CH2 HN
O
C CH2 CH2 HN
O
C CH
CH3
CH3
O
O
OP
O
NH2
N
N
N
N
O
O
O
P O CH2
OH
O
cysteamin
beta-alanin
Pantothenin
acid
Acyl side
Biochemistry-3
74
S-Adenosylmethionine (SAM)
• "Active methyl"
• S with 3 bonds
• cofactor methylation
• enzymes for example: phosphatidylcholine
phosphatidylethanolamine →
methionine from homocysteine arises
Biochemistry-3
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S-Adenosylmethionin (SAM)
N
N
N
N
NH2
O
OHOH
S
CH3
HOOC CH
NH2
CH2CH2
Biochemistry-3
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Tetrahydrofolate
• Transport (C1) fragments
- bind to the nitrogens N5 and / or N10
- purine biosynthesis, methylation uracil
- C1 fragments of the redox state:
• reduced: methyl -CH3
• Mild reduced: methylen -CH2•
oxidized:
formyl -CHO formimino -CH=NH
methenyl -CH= Biochemistry-3
77
Sources C1 residues
• Trp catabolism: formate → formyl
His catabolism: formimino → methenyl
Catabolism Ser: hydroxymethyl methylene →
Catabolism of Gly: methylene
Methionine → SAM → methyl + homocysteine
Biochemistry-3
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Tetrahydrofolate
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 15
Transport C1.................
105N
N
N
NH2N
OH
CH2NH C NH
O
CHCH2CH2COOH
COOH
H
H
Biochemistry-3
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Vitamin B12 cyanocobalamine and/or
hydroxocobalamine
http://www.chm.bris
.ac.uk/motm/vitami
nb12/b12.gif
Biochemistry-3
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Cofactor is methylcobalamin
• methylation reactions
• remethylation of homoCys to
Met
• hydroxocobalamin - treatment of
cyanide poisoning
binds to toxic cyanide anions
cyanocobalamin intravenous infusion
http://www.methylcobalamininfo.com/wp-
content/uploads/2013/08/methylcobalamin2.png
Biochemistry-3
81
Two reactions with B12
• S-methylation of homocysteine = regeneration,
methyl is withdrawn from methyl tetrahydrofolate
(and thereby creating H4 folate)
• propionyl-CoA  succinyl-CoA
B12 necesary for regneration of
tetrahydrofolate
Biochemistry-3
82
Thiamin is vitamin B1
• cofactor is thiamindiphosphate (TDP)
oxidative decarboxylation of pyruvate,
• 2-oxoglutarate
transfers so. activated aldehyde
pyruvate  acetyl-CoA
• 2-OG → succinyl-CoA (CC)
N
NH3C NH2
N
S
CH3
CH2CH2OH
Biochemistry-3
83
Thiamindiphosphate (TDP) is cofactor of
oxidative decarboxylation of pyruvate
glucose  pyruvate  acetyl-CoA
CC
TDP
Binding of pyruvate for
decarboxylation
N
N
N
SH3C NH2
CH3
CH2CH2 O P
O
O
O P
O
O
O
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 16
Biochemistry-3
84
The mechanism of enzyme
reactions, metalloenzymes,
kinetics, activity, enzymes in
medicine
Biochemistry-3
85
Active site of enzyme
• small portion of the
molecule,
• the three-dimensional
arrangement
deep slot (eg. amylase),
surface depression
• Attending side chains
AA distant in the
primary structure
• Protein flexibility allows
adaptation induced
conformation
corresponding substrate
Biochemistry-3
86
Binding of substrate
• substrate binding to the active site causes
conformational change of the molecule
corresponding to the enzyme - induced
adaptation
creates a complex enzyme-substrate
E + S ES E + P
Biochemistry-3
87
catalytic groups
• the realization of chemical transformations in the
active site are used so. catalytic groups:
- nucleophilic (cysteine sulfhydryl, serine OH)
- acidic (Asp, Glu), - basic (His, Arg, Lys)
• acid-base (proton transfer)
transient covalent bond
metal ion catalysis (metalloenzymes)
electrostatic interactions (without water)
deformation of the substrate
Possible mechanisms
Biochemistry-3
88
Example: the active site of chymotrypsin? Nucleophilic attack of
OH to the carbonyl carbon of serine peptide bond - serine
protease
N C
O
H
His
N
N
H
Ser
OH
195
57
NOVÁK, Jan. Biochemie I.
Brno: Muni, 2009, Enzymy s.
19
Biochemistry-3
https://www.youtube.com/watch?v=gJNMryCX3YY
89
The active site of chymotrypsin: cleavage of
peptide bonds
His
N
N H
Ser
O
C
O
NH2
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 19
Biochemistry-3
90
Metaloenzymes
• containing functional metal ions, which participate
directly catalyzed reactions, metal ions are bound
quite tightly (Enz-M)
some enzymes need metal ions only to activate, in
which case they are bound weakly (Enz ... M), a
bivalent metal ions, Ca2 + (coagulation factors),
Mg2 + (kinase)
Biochemistry-3
91
Metal cation is a part of the ternary complex
• three components form a complex: enzyme (Enz), a
substrate (S) and the metal cation (M)
various types (bridged) complexes Enz-SM, Enz-MS
sometimes arise cyclic complexes
• to vacant orbitals can accept an electron pair to form a nucleophile-
binding
may form chelates with appropriate groups of the enzyme or substrate
structure deformation tension which facilitates chemical
transformation
coordination sphere of metal acts as a three-dimensional template
stereospecific control
mechanisms
Biochemistry-3
92
molybdenum
• Parts of some oxidoreductases
Part of the cofactor - molybdopterin
Xanthine oxidase (xanthine  uric acid)
Sulfitoxidasa (HSO3
-  SO4
2-)
Aldehydoxidasa (less common, acetaldehyde to acetic acid.)
Sources Mo: legumes, whole grains
Biochemistry-3
93
Zinc
• Many enzymes
• Alcoholdehydrogenase (ethanol  acetaldehyd)
• carbonatedehydratase (H2O + CO2  H2CO3)
• carboxypeptidase (cleavage of the polypeptide
from the C-terminus)
• Cu, Zn-superoxiddismutase (cytosolic izoform)
(2 •O2
- + 2 H+  O2 + H2O2)
Sources Zn: red meat, shellfish, legumes,
sunflower and pumpkin seeds, whole grain
cereals Biochemistry-3
94
Copper
• oxidoreductases
• Ceruloplazmin (ferrooxidase) (Fe2+  Fe3+)
• Cytochrom-c-oxidase (RCh, transport of e- to O2)
• Monoaminooxidase (MAO, inactivation of biogenic
amines, H2O2,
• Dopaminhydroxylase (dopamin  noradrenalin)
• Lysyloxidase (colagene, Lys  alLys)
Cu Sources: liver, meat, cocoa, legumes, nuts
Biochemistry-3
95
Mangan
• Numerous hydrolases, decarboxylase
transferase
Mn-superoxide dismutase (mitochondrial isoform)
• Arginase ((Arg  urea + ornithine)
• The synthesis of proteoglycans, glycoproteins
Sources Mn: legumes, whole grains, nuts
Biochemistry-3
96
Iron
• Heme enzymes, non-heme transporter
• Catalase (hem, H2O2  H2O + ½O2)
• Myeloperoxidase (hem, neutrophil)
H2O2 + Cl- + H+  HClO + H2O
• Cytochromes (heme electron carrier in the RCh)
• Fe-S proteiny (nehem, přenos elektronů v DŘ)
Sources Fe products from pork, goose, duck blood
(red) meat, liver, egg yolk, nuts
Biochemistry-3
97
Selen
• Several enzymes (redox reaction), Se is always
selenocysteine
• glutathione peroxidase
(2 GSH + H2O2  2 H2O + G-S-S-G)
• Dejodasy thyronin (thyroxin T4  trijodthyronin T3)
• Thioredoxin reductase (ribose  deoxyribosa)
• Selenoprotein P (plasma, antioxidant function?)
Sources Se: cephalopods, marine fish, legumesBiochemistry-3
98
Basic concepts of kinetics
• reaction: S  P (S = substrate, P = product)
• definition of reaction rate:
]
l.s
mol
[0
]P[]S[







tt
v
Note: this is defined as the average reaction rate,
instantaneous velocity: d [S] / dt (derivative, share two infinitely
small numbers) Biochemistry-3
99
What determines the rate of
reaction?
• the substrate concentration [S]
temperature
in the presence of effector (catalyst
inhibitor)
In addition, enzymatic reactions:
enzyme concentration [E]
pH
Biochemistry-3
100
The kinetic equation for the reaction
S  P
v = k [S] = k [S]1  reaction of
1. order
k = rate constant
v
[S]
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 21
Biochemistry-3
101
The substrate concentration during the
reaction decreaseskinetic
curve
[S]
t
speed is measured from the
kinetic curves
NOVÁK, Jan. Biochemie
I. Brno: Muni, 2009,
Enzymy s. 21
Biochemistry-3
u enzymových reakcí pouze v
laboratorních podmínkách
v
[S]
102
Reaction 0. order is special case
• the reaction rate doesnt depends on the concentration of the
substrate
• v = k [S]0 = k . 1 = k = constant
• occurs when a large excess of S,
• so that the loss is almost negligible
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 22
on laboratory condition
Biochemistry-3
103
The initial rate vo
• speed measured before significant amounts of
product formed
• the highest speed value
• "Virtual value„
• is not affected by the loss of substrate
conversion or return the product
• sets of curves,
Biochemistry-3
104
Dependence of vo on concetration of substrate
• Michaelis-Menten equation
• single-substrate reaction
m
o
K
Vv


]S[
]S[
max
Vmax = maximal speed (for one concetration of enzyme)
Km = Michaelis constantBiochemistry-3
105
Saturation curve - Michaelis-Menten equation
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 23
Km
mol/l[S]
vo
Vmax
enzym is fully
ocupied with
substrate
Vmax
2
Biochemistry-3
106
For [S] << Km
1max
max ]S[]S[
]S[
]S[
k
K
V
K
Vv
mm
o 


At low substrate concentrations the
reaction is governed by 1st order
kinetics
Biochemistry-3
107
For [S] >> Km
0
maxmaxmax ]S[
]S[
]S[
]S[
]S[
kVVVv
m
K
o 


At high substrate concentrations, the
reaction is governed by the kinetics of
the 0th order
Biochemistry-3
108
Two parts of saturation curve
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 23
mol/l[S]
vo
kinetics 0. order
kinetics
1. order
Enzyme is saturated by
substrate
Biochemistry-3
109
For [S] = Km
2]S[2
]S[
]S[]S[
]S[ max
maxmax
V
VVvo 


Biochemistry-3
110
Biochemical importance of Km
• substrate concentration at which the reaction
has half of the maximum speed
• at this concentration the enzyme is saturated
with 50%
• Km has the dimension of concentration (mol / l)
• Km is inversely proportional to the affinity of
the enzyme for the substrate,
• if there is more structurally similar substrates,
which has the smallest Km is considered to be
the most natural for the enzyme
Biochemistry-3
111
How to obtain a saturation
curve?
• set of experiments, a constant
concentration of the enzyme, various
concentrations of the substrate ranges
from 2 to 3 orders
• from kinetic curvs is estimated vo
• vo is graphically plotted against the
relevant [S]
• arises hyperbolic saturation curve
Biochemistry-3
112
Distinguish
Kinetic curve
• Time record of one
reaction
• [S] = f (t)
Saturation curve
• dependence obtained
from many of the
same reactions
• vo = f ([S])
[S] …….. concetration of substrate
f …………function
t …… time
vo ……….. Initial speed
Biochemistry-3
113
Value Vmax a Km characteristics of kinetic
properties of enzymes
• is easily determined from the graph of the
linearized
Lineweaver-Burk
double reciprocal plot
• 1/vo against 1/[S]
Biochemistry-3
114
m
o
K
Vv


]S[
]S[
max









]S[]S[
]S[1
]S[
]S[11
maxmax
mm
o
K
V
K
Vv
]S[
111
maxmax

V
K
Vv
m
o
Biochemistry-3
115
Reciprocal relationship is the equation of a
line
]S[
111
maxmax

V
K
Vv
m
o
1/vo ................ the dependent variable
1/[S] ............... independent variable
Km/Vmax ........... slope of the line
1/Vmax .............
the intercept of the dependent variableBiochemistry-3
116
Linear graph: 1/vo is function of 1/[S]
1/vo
1/[S]
1 / Vmax
- 1 / Km
NOVÁK, Jan.
Biochemie I. Brno:
Muni, 2009, Enzymy s.
24
Biochemistry-3
117
The concentration of enzyme [E] also affects the
speed
saturated enzyme : vo = k [E]t
• [E]t is the total concentration of enzyme
[E]t = [E] + [ES]
Biochemistry-3
118
Saturation curves for different
concentrations of enzyme
vo
[S]
[E1]
[E2] > [E1]
[E3] > [E2]
KM is unchanged, Vmax increases
NOVÁK, Jan. Biochemie I. Brno:
Muni, 2009, Enzymy s. 25
Biochemistry-3
119
How to determine the amount of enzyme
in biological material?
• very difficult
• low (trace) the concentration of
enzyme
• present in many other proteins
• normal chemical reactions are not
applicable
• not specific for differentiation of
individual enzymes
Biochemistry-3
120
The amount of enzyme in the biological material
can be determined in two ways
Indirect determination
Catalytic concetration
• μkat/l
• determined by the
product of the enzyme
reaction
most clinically important
enzymes
Direct determination
• mass concentration
• μg/l
• determine the enzyme
molecule as antigen
(immunoassay)
• some, for example. tumor
markersBiochemistry-3
121
The catalytic activity of the
enzyme
• Unit katal, 1 kat = mol/s
• One katal is the catalytic activity of the enzyme at which the
reaction is converted per mole of substrate for second
IU (international unit)
1 IU = μmol/min
1 μkat = 60 IU
1 IU = 16,6 nkat
Biochemistry-3
122
Catalytic enzyme concentration
• Activity is based on the volume of biological
fluid (blood serum)
• Units mkat/l, kat/l
Biochemistry-3
123
Compare the different analytical approaches
Glucose
• substrate
• low molecular weight
concentration in
serum
• 3,3-5,6 mmol/l
• Glucose is
determined directly
ALT
• enzyme
high molecular weight
cat. concentration in serum
• 0,2-0,9 kat/l
• determined not by the
enzyme, but the product or a
cofactor in enzymatic
reactions
Biochemistry-3
124
Methodology for determination of ALT (all ingredients are colorless)
optický test
Semináře, s.18
Alanine + 2-Oxoglutarate
PyridoxalP
Pyruvate + Glutamate
Blood serum
(obsah. ALT)
LD
Lactate + NADPyruvate +NADH + H
Decreace of absorbance
A/t
Reaction components
Sample with EN
Biochemistry-3
125
Determination of the catalytic activity in
laboratory
• optimal conditions (temperature, pH, cofactors)
• measured [S] or [P] in a certain time interval
• kinetics of the 0th order, [S] >> Km -----
• saturated enzyme, velocity is constant,
• Approaching Vmax
Biochemistry-3
126
Two methods for the determination of
catalytic concentration of enzymes
• Kinetic
• continuously measured
[S] or [P] (eg., after 10
s)
• Plot the kinetic curve
it is found in the
kinetic curves
• accurate method
constant time/End
point
• measured [P]
after the
reaction
• kin. curve is not
needed
• average speed
• [P]/ t
• Less accurate
methodBiochemistry-3
127
Enzyme inhibition (reduction of
activity)
irreversible
inhibitor tightly bound to the
enzyme (active place)
organophosphates
heavy metal ions
cyanides
reversible
loosely bound inhibitor
balance E + I …. E-I
inhibitor can be removed (dialysis,
gel. filtration)
two basic types: competitive,
noncompetitive
Biochemistry-3
128
competitive inhibition
• inhibitor is structurally similar to the
substrate
•
binds to the active site
•
competes with the natural substrate
binding site
Biochemistry-3
129
Natural substrae x competitive inhibitor
Malonate is inhibitor of
succinatedehydrogenase
http://www.s-
cool.co.uk/assets/test_its/
alevel/biology/biological
-molecules-and-
enzymes/quest24.jpg
Biochemistry-3
CH3OH H COOH
alcoholdehydrogenase
CH3CH2OH
130
Methanol poisoning is treated with ethanol
Formation of toxic
products is inhibited
by ethanol, methanol,
which is displaced
from the binding site
of the enzyme competitive
inhibition
dehydrogenation of
methanol
The toxicity of methanol lies not so much
in the action of this substance (even
cause CNS depression), but rather in the
behavior of its breakdown products formaldehyde
and formic acid. [11]
Methanol itself is metabolized roughly half
the rate of the ethanol. The total
elimination of methanol is slow,
corresponding to roughly one-seventh the
speed for ethanol. Moreover, ethanol has
about twenty times higher than the affinity
for alcohol dehydrogenase methanol,
therefore, the preferred substrate. This
allows administered ethanol (also
fomepizole) as antidote for poisoning
because it significantly slows down the
metabolism of methanol, and thus
significantly reduce its biochemical and
clinical effects.
Biochemistry-3
Km
mol/l[S]
vo
Vmax
Km inh 131
Competitive inhibition
• maximum speed is reached at higher value [S]
• Vmax no change
• Km increase
NOVÁK, Jan. Biochemie I.
Brno: Muni, 2009, Enzymy s. 29
Biochemistry-3
132
Competitive inhibition
1/vo
1/[S]
1 / Vmax
- 1/Km - 1/Km
1 / Vmax
Biochemistry-3
133
Noncompetition inhibition
• The inhibitor binds outside the active center on the complex E and ES
• Km does not change (the active site is free for substrate)
• Vmax is reduced because the concentration decreases functional
complex
Km
mol/l[S]
vo
Vmax
Km inh
Vmax inh
NOVÁK, Jan. Biochemie
I. Brno: Muni, 2009,
Enzymy s. 29
Biochemistry-3
134
Non competitive inhibition
1/vo
1/[S]
1 / Vmax
- 1 / Km
- 1 / Km
1 / Vmax inh
Biochemistry-3
135
Many drugs are enzyme inhibitors
• Acetylsalicylic acid (cyclooxygenase)
• Ibuprofen (cyclooxygenase)
• Statins (HMG-CoA reductase) - lipid lowering drugs reduce
cholesterol synthesis (lovastatin)
• ACE inhibitors (angiotensin converting enzyme) - treatment of
hypertension (enalapril)
• Reversible inhibitor of acetylcholinesterase (neostigmine) neuromuscular
disease, postoperative intestinal atony
• Selective brain acetylcholinesterase inhibitors (rivastigmine,
galantamine) - Alzheimer's disease
Biochemistry-3
136
Antibiotics inhibit the enzymes necessary
for a life of bacteria
• Penicillin - inhibit transpeptidasy
(construction of cell walls)
• Tetracyclines, macrolides, chloramphenicol inhibition
of protein synthesis
• Fluorinated quinolones (ciprofloxacin) inhibition
of bacterial gyrase (topoisomerase
II) (untwisting of DNA during replication)
Biochemistry-3
137
1. Control of the number of molecules of the
enzyme
2. Regulation of the biological activity of the
enzyme
3. Availability and concentration of substrate and /
or cofactor (in vivo less significant factor)
Regulation of enzyme activity
(three general ways)
Biochemistry-3
138
Regulating the amount of
enzyme
• Controlled constitutive enzyme, protein
synthesis and induce the expression of genes,
speed regulation of transcription, posttranscriptional
modifications of RNA,
regulation of the speed of translation and
posttranslational modifications
• Controlled degradation enzyme specific
intracellular proteases - determine the
different biological half-lives of enzymes
Biochemistry-3
139
Regulation of the biological activity of the
enzyme
• Isoenzymes (one type of response is
regulated differently in various tissues)
• Activation of the enzyme and irreversible
partial proteolysis
• Breakage of covalent modification of the
enzyme
• allosteric regulation
Biochemistry-3
140
Isoenzymes
• catalyze the same reaction, but differ in primary
structure and thus phys.-chem. and kinetic
properties
• often have different tissue distribution
• They are determined by electrophoresis
• isoforms - a more general term (include more
pseudoizoenzymy, posttranslational variants)
Biochemistry-3
141
Creatinkinase (CK) is dimer and form isoenzymes
Izoenzyme % of activity increased
CK-MM muscle 94-96% muscle trauma
CK-MB heart to 6% heart attack
CK-BB Brain trace brain Injury
In the cells, the "cytosolic" CK enzymes consist of two subunits, which can be either
B (brain type) or M (muscle type). There are, therefore, three different isoenzymes:
CK-MM, CK-BB and CK-MB. The genes for these subunits are located on different
chromosomes: B on 14q32 and M on 19q13. In addition to those three cytosolic CK
isoforms, there are two mitochondrial creatine kinase isoenzymes, the ubiquitous
and sarcomeric form. The functional entity of the latter two mitochondrial CK
isoforms is an octamer consisting of four dimers each.[4]
While mitochondrial creatine kinase is directly involved in formation of phosphocreatine
from mitochondrial ATP, cytosolic CK regenerates ATP from ADP, using
PCr. This happens at intracellular sites where ATP is used in the cell, with CK acting
as an in situ ATP regenerator.
Biochemistry-3
142
Activation of the enzyme by partial proteolysis
• Active enzyme arises irreversible cleavage of a particular sequence
of the proenzyme molecule
• Proteinase in GIT ( pepsinogen pepsin)
• Blood clotting factors
• Proteases (caspases) activated during apoptosis
Biochemistry-3
143
Reversible covalent modification of the
enzyme
• phosphorylation catalyze kinase -PO3 2phosphoryl
transfer from ATP to the OH group of
the enzyme (Ser, Thr, Tyr)
• reciprocal plot (dephosphorylation) phosphatase
catalyze the hydrolysis of ester-bound phosphate
• Other modification: carboxylation,
acetylation……………….
Biochemistry-3
144
Phosphorylation and dephosphorylation of
enzyme
Enzym Serin OH Enzym Serin O P
O
O
O
ATP ADP
H2O
P
O
O
OHO
kinase
phosphatase
Biochemistry-3
145
Mechanism of phosphorylation
http://web2.mendelu.cz/af_291_pro
jekty2/vseo/stranka.php?kod=1722
Biochemistry-3
146
Examples
glycogen phosphorylase
Catalyzes the cleavage of
glycogen by inorganic.
Phosphate
Phosphorylated enzyme is
active
Dephosphorylated enzyme is
inactive
Glykogensynthasa
Catalyzes the synthesis of
glycogen from UDP-glucose
The phosphorylated enzyme is
inactive
Dephosphorylated enzyme is
active
Biochemistry-3
147
Allosteric enzymes are oligomeric
• multiple subunits often regulatory and catalytic
• the enzyme binds effector structurally distinct from
the substrate often product
• binds to the allosteric sites - other than the active
site
• binding causes a conformational change in the
enzyme activity …. change - allosteric activation or
inhibition
Biochemistry-3
148
Saturation curve of allosteric enzymes
are sigmoid
bez
efektoru
NOVÁK, Jan. Biochemie I.
Brno: Muni, 2009, Enzymy s. 32
[S]
vo activation
inhibitionWithout
effector
Biochemistry-3
149
Cooperative effect
• for oligomeric enzymes and proteins
• more subunits --- more binding sites
• binding of substrate (or other
substance) to one subunit induces a
conformational change in the other,
that bind other molecules more easily
(difficult)
• Example: hemoglobin × myoglobin
Biochemistry-3
150
Using of enzymes in medicine
1. enzymes as indicators of a pathological condition: when
cell damage increases intracellular enzyme activity in the
extracellular fluid
2. enzymes as analytical reagents in her lap. biochemistry
3. enzymes as medicaments
Biochemistry-3
151
Examples of enzymes in clinical diagnosis
a The serum values ​​for men over 15 years
ALT alanine aminotransferase, creatine kinase CK,
PSA prostate specific antigen, neuron specific enolase NSE
Enzym e Referenční hodnoty Interpretace zvýšení
ALT
(alaninaminotransferase)
to 0,9 μkat/l hepatopatie
CK
(creatinkinase)
to 4 μkat/l myopatie, heart atact
PSA
(prostatic specific antigene)
to 4 μg/l carcinom of prostate
Biochemistry-3
152
Enzymes as analytical reagents
Enzyme Origine of enzyme The substance / method
Glucosaoxidase Aspergillus niger glucose
Peroxidase křen (Armoracia sp.) glucose
Lipase Candida sp. triacylglycerol
Cholesteroloxidase Pseudomonas sp. cholesterol
Uricase Candida sp. Uric acid
Bilirubinoxidase Myrothecium sp. bilirubin
Urease bob (Canavalia sp.) urea
Lactatdehydrogenasa Pediocus sp. ALT, AST
Taq polymerase Thermus aquaticus PCR
Biochemistry-3
153
Enzymatic determination of
glucose
glucose + O2  gluconolacton + H2O2
H2O2 + H2A  2 H2O + A
glucosaoxidasa
peroxidase
chromogen Product (absorbance)
Principle of glucose analyzers
Biochemistry-3
154
Personal glucometers
• intended for personal control diabetic
• glucose oxidase is anchored on the solid phase
• H2O2 produced is determined by another method (using Pt
electrodes)
• the display shows the concentration of glucose (mmol / l)
Biochemistry-3
155
Personal glucometers
http://zdrav-pro.cz/wp-
content/uploads/glukometr-
g423-1.2.jpg
Biochemistry-3
156
Pancreatic enzymes in therapy
• a mixture of enzymes (lipases, amylases,
proteases) obtained from porcine pancreas
• indication: pancreatic secretory insufficiency
of different etiology, cystic fibrosis
• dosage: 3 times a day with meals
• range of OTC products
acid-resistant capsules to
disintegrate in the duodenumBiochemistry-3
157
Asparaginase in the treatment of
leukemia
• Catalyze hydrolysis of the amide group asparagine
• Asn + H2O  Asp + NH3
• L-asparagine is essential for protein synthesis of some
tumor cells
• Hydrolysis of Asp leads to a reduction of proliferation
• Indications: acute lymphoblastic leukemiaKatalyzuje
hydrolýzu amidové skupiny asparaginu
Biochemistry-3
158
fibrinolytic enzyme
• drugs that dissolve blood clots
• streptokinase (bacterial), urokinase (human)
• cleaves plasminogen to plasmin - that causes degradation
of fibrin and thrombolysis
• indications: venous thrombosis, pulmonary embolism,
acute IM
Biochemistry-3
159
Proteases in local therapy
• fibrinolysin, chymotrypsin, collagenase
• after topical application leads to lysis of necrotic
tissue, do not harm healthy cells (containing
protease inhibitors)
• main indication in surgery
• festering wounds, venous ulcers, diabetic
gangrene, pressure ulcers, postoperative wounds,
etc.. Biochemistry-3
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Proteases in systemic therapy (oral
administration)
• Trypsin, chymotrypsin, plant proteases - papain (papaya),
bromelain (pineapple)
• Some studies suggest an anti-inflammatory effect, influencing
immunity in autoimmune disease
• Indication: auxiliary drug for rheumatoid arthritis, traumatic
inflammation and edema, lymphoedema, phlebitis, etc..
• CHC, quite expensive (Wobenzym, Phlogenzym etc..) to 30
tabl. daily
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Example 1
When the enzymatic reaction of a substrate
solution was added to the buffer containing
the enzyme is added to the sample (0.1 ml).
After 5 min was determined 0.2 mmol of
product.
What is the catalytic concentration of
enzyme in the sample?
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Example 1 - Solution
t = 5 min = 5. 60 s = 300 s
for 300 s … 0.2 mmol of product formed
for 1 s … x = 0,2/300 = 6,7 .10-4 mmol / 0,1 ml sample
for 1 litr sample = 6,7 . 10-4 . 104 = 6,7 mmol/l.s = 6,7 mkat/l
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Example 2
The reaction mixture contained:
2.5 ml buffer
0.2 ml of coenzyme NADH (optical test)
0.1 ml of blood serum
0.2 ml of substrate solution
For 60 s was a decrease in absorbance of coenzymeA
= 0,03. NADH = 6220 l/mol.cm, L = cuvette
width of 1 cm. What is catalytic concentration
of enzymes? Biochemistry-3
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Solution of example 2
Serum was diluted : Vcon/Vorig = 3,0 / 0,1 = 30
Lambert-Beer: A =  c l / for t 
60 s:
Dilution: 30 . 8 .10-8 = 2,4 . 10-6 mol/l.s =
2,4 . 10-6 kat/l = 2,4 kat/l
mol/l.s10.8
6016220
03,0 8







tl
A
t
c

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catalytic enzyme concentration = chemical
reaction rate
[mol / l.s]
Biochemistry-3