Enzymes I
General features, cofactors
 Department of Biochemistry, FM MU, 2013 (J.D.)
1
Literature for Biochemistry I
•
Lecture files on is.muni.cz.
•
Tomandl J., Táborská E.: Biochemistry I – Seminars. MU, 2012
•
Harvey R.A., Ferrier D.R.: Biochemistry. 5th ed.,
Lippincott Williams & Wilkins, 2011.
•
Koolman J., Röhm K.H.: Color atlas of biochemistry,
Thieme, 2013.
2
General features of enzymes
CAUTION: peptidyltransferase is ribozyme
• biocatalysts
(AK)n-tRNA1 + AK-tRNA2
rRNA
 tRNA1 + (AK)n+1-tRNA2
• different types of proteins / also RNA (ribozyme)
with covalently attached prosthetic group and/or metal cation,
oligomeric / multienzyme complexes / associated with membranes etc.
• different distribution in cell and in the body, make isoforms (isoenzymes)
• specific (towards substrate and reaction), highly effective
• work under mild conditions
• in vivo - can be regulated in two ways (activity of enzyme, quantity of enzyme)
• in vitro - sensitive to many factors
3
Enzymes are highly efficient catalysts
• decrease activation energy  increase the reaction rate
• much more efficient than other (inorganic) catalysts
• remain unchanged after reaction
• do not alter equilibrium constant K
• in vitro sensitive to many factors
4
Enzymes work under mild conditions
• narrow temperature range around 37 °C
• over 50 °C become denaturated = inactivated
• narrow pH range  pH optimum
• most intracellular enzymes have pH optima around 7
• digestion enzymes function in rather stronger acidic / alkaline
environment (pepsin 1-2, trypsin ~ 8)
5
Dual specifity of enzymes towards:
Reaction
Substrate
catalyze just
work with one substrate
one type of reaction
(or group of similar substrates)
often stereospecific
6
Enzymes are stereospecific catalysts
there are two types of stereospecific conversions:
1. non-chiral substrate  chiral product(one enantiomer)
pyruvate  L-lactate
fumarate  L-malate
2. chiral substrate(one enantiomer)  product
L-alanine  pyruvate
(D-alanine does not react)
D-glucose   pyruvate (L-glucose does not react)
chiral signal molecule  complex with receptor  biological response
chiral drug(ant)agonist  complex with receptor  pharmacological response
7
1. non-chiral substrate  chiral product
Hydrogenation of pyruvate
when pyruvate is hydrogenated without enzyme (in vitro),
reaction product is the racemic mixture of D-lactate and L-lactate:
H
O
C
HO
C
COOH
CH3
HO
O
C
H + H
CH3
H
COOH
C
OH
CH3
L-laktát
L-Lactate + D-laktát
D-Lactate
in reaction catalyzed by lactate dehydrogenase (in vivo),
pyruvate is reduced stereospecifically to L-lactate only:
H
O
HO
C C
CH3
O
COOH
HO C H
CH3
Enzyme
L-Lactate
8
1. non-chiral substrate  chiral product
Non-enzymatic hydration of fumarate
COOH
addition
from
adice z levé
one
side
strany
O
HO C H
CH2COOH
L-malát
L-malate
H COOH
H
H
C
C
HOOC H
H
O
addition
from
adice
z pravé
another
strany side
COOH
H C OH
H
CH2COOH
D-malát
D-malate
fumarate
in vitro reaction proceeds to racemic D,L-malate
9
1. non-chiral substrate  chiral product
Enzymatic hydration of fumarate (citrate cycle)
H
O
H
reagent
H C C COOH
HOOC
H
Substrát
substrate
Enzym
enzyme
in vivo just one enantiomer (L-malate) is produced
10
1. non-chiral substrate  chiral product
Hydrogenation of D-fructose in vitro gives two epimers
reaction site is planar
CH2OH
CH2OH
C O
HO C H
CH2OH
H C OH
2H
HO C H
HO C H
+
HO C H
H C OH
H C OH
H C OH
H C OH
H C OH
CH2OH
CH2OH
CH2OH
D-glucitol
D-mannitol
D-fructose
H C OH
in vivo: enzymatic reaction gives just one product (D-glucitol)
11
2. chiral substrate  product
Enzymes or receptors recognize only one enantiomer
If the reactant of enzymatic reaction is a chiral compound,
only one of two enantiomers is recognized as the specific substrate.
Chiral substrates/signal molecules are bound to the stereospecific
enzymes/receptors at three sites:
R
see also
MCH II, p. 13
R
x
proper enantiomer
X
not-fitting enantiomer
12
Enzyme nomenclature: the ending -ase
Systematic names identify the enzymes fully with the EC code number,
contain information about substrate and type of reaction,
not very convenient for everyday use.
Recommended (accepted) names are shorter than systematic names,
include also some historical names (pepsin, amylase)
EC (abbr. Enzyme Commission) of International Union of Biochemistry (IUB)
major class number
. subclass number
. sub-subclass number
. enzyme serial number
http://www.chem.qmul.ac.uk/iubmb/enzyme/
13
Examples of enzyme names
Recommended name: alcohol dehydrogenase
Systematic name: EC 1.1.1.1 ethanol:NAD+-oxidoreductase
Reaction: ethanol + NAD+  acetaldehyde + NADH + H+
Recommended name: alanine aminotransferase (ALT)
Systematic name: EC 2.6.1.2 L-alanine:2-oxoglutarate-aminotransferase
Reaction: L-alanine + 2-oxoglutarate  pyruvate + L-glutamate
14
Classification of enzymes: six classes according to reaction type
(each class comprises other subclasses)
Enzyme class
General scheme of reaction
1. Oxidoreductases
Ared + Box  Aox + Bred
2. Transferases
A-B + C  A + C-B
3. Hydrolases
A-B + H2O  A-H + B-OH
4. Lyases
A-B  A + B
5. Isomerases
A-B-C  A-C-B
6. Ligases (synthetases)
A + B + ATP  A-B + ADP + Pi
(reverse reaction: synthases)
15
1 Oxidoreductases
catalyze the oxidation or reduction of substrate
subclasses:
• dehydrogenases catalyze the transfers of two H atoms
• oxygenases catalyze the incorporation of one/two O atoms
into the substrate (monooxygenases, dioxygenases)
• oxidases catalyze transfers of electrons between substrates
(e.g. cytochrome c oxidase, ferroxidase)
• peroxidases catalyze the decomposition of peroxides
Example: lactate + NAD+  pyruvate + NADH + H+
Recommended name: lactate dehydrogenase
Systematic name: (S)-lactate:NAD+ oxidoreductase
16
2 Transferases
catalyze the transfer of a group from one to another substrate
subclasses:
• aminotransferases, methyltransferases, glucosyltransferases
• kinases phosphorylate substrate by the transfer
of phosphoryl group PO32– from ATP (e.g. hexokinases, protein kinases)
Example: glucose + ATP  glucose 6-P + ADP
Recommended name: glucokinase
Systematic name: ATP:D-glucose phosphotransferase
17
Example: Phosphorylation of glucose
O
HO
O
O OH
+ ATP
OH
O
P
O
O OH
glucokinase
glukokinasa
OH
+ ADP
OH
OH
OH
glucose
glukosa
OH
glucose
6-phosphate
glukosa-6-fosfát
18
3 Hydrolases
catalyze the hydrolytic splitting of esters, glycosides, amides, peptides etc.
subclasses:
• esterases (lipases, phospholipases, ribonucleases, phosphatases)
• glycosidases (e.g. sucrase, maltase, lactase, amylase)
• proteinases, peptidases (pepsin, trypsin, cathepsins, caspases/apoptosis,
dipeptidases, carboxypeptidases, aminopeptidases)
• amidases (glutaminase, asparaginase)
• ATPases (split anhydride bonds of ATP)
Example: glucose 6-P + H2O  glucose + Pi
Recommended name: glucose 6-phosphatase
Systematic name: glucose 6-phosphate phosphohydrolase
19
Example: glucose 6-phosphatase
O
O
O
P
HO
O
O OH
O OH
+
OH
OH
H2O
OH
glukosa-6-fosfát
glucose 6-P
P
+
O
OH
OH
O
O
OH
glucose
glukosa
20
OH
Compare two antagonistic enzymes
ATP
ADP
kinase
O
Enzym Serin OH
Enzym Serin O P O
O
O
O P OH
phosphatase
H2O
O
21
Glutaminase is amidase which catalyzes
the deamidation of glutamine
COOH
COOH
H2N CH
H2N CH
CH2
CH2
O
C
NH2
glutamin
glutamine
H2O
CH2
glutaminase
CH2
O
C
+ NH3
OH
glutamate
glutamát
22
ATPase catalyzes the exergonic hydrolysis
of phosphoanhydride bond in ATP
ATP + H2O  ADP + Pi + energy
Example: muscle contraction
myosine head exhibits ATPase activity,
chemical energy of ATP is transformed into
mechanical work (actin-myosin contraction)
23
Examples of lysosomal hydrolases
Hydrolase
Bond hydrolyzed
Glucosidase
Galactosidase
Hyaluronidase
Arylsulfatase
Lysozyme
Cathepsin
Collagenase
Elastase
Ribonuclease
Lipase
Phosphatase
Ceramidase
glycoside
glycoside
glycoside
sulfoester
glycoside
peptide
peptide
peptide
phosphodiester
ester
phosphoester
amide
24
Distinguish: lysozyme × lysosome
Lysozyme is enzyme
• compound word, lyso (Greek lysis) + zyme (from enzyme)
• hydrolase, glycosidase, cleaves β-1,4-glycoside bond
in bacterial heteropolysaccharides, antiseptic defense
• occurs in saliva, tears, and other body fluids
Lysosome is intracellular digestion organelle
• Greek compound word from lysis (to lyse) and soma (body)
• typical for animal cells
• acidic pH, contains many acidic hydrolases
25
4 Lyases
catalyze non-hydrolytic splitting or forming bonds C–C, C–O, C–N, C–S through
removing or adding, respectively, a small molecule (H2O, CO2, NH3)
Some frequent recommended names:
• ammonia lyases (e.g. histidine ammonia lyase: histidine  urocanate + NH3)
• decarboxylases (amino acid  amine + CO2)
• aldolases (catalyze aldol cleavage and formation)
• (de)hydratases (e.g. carbonate dehydratase: CO2 + H2O  H2CO3)
Example: fumarate + H2O  L-malate
Recommended name: fumarate hydratase
Systematic name: (S)-malate hydro-lyase (fumarate-forming)
26
5 Isomerases
catalyze intramolecular rearrangements of atoms
examples:
• epimerases
• racemases
• mutases
Example: UDP-glucose  UDP-galactose
Recommended name: UDP-glucose 4-epimerase
Systematic name: UDP-glucose 4-epimerase
27
6 Ligases
catalyze the formation of high-energy bonds C–C, C–O, C–N
in the reactions coupled with hydrolysis of ATP
Frequent recommended names:
carboxylases
synthetases
(e.g. glutamine synthetase: glutamate + ATP + NH3  glutamine + ADP + Pi)
Example: pyruvate + CO2 + ATP + H2O  oxaloacetate + ADP + Pi
Recommended name: pyruvate carboxylase
Systematic name: pyruvate:carbon-dioxide ligase (ADP-forming)
28
!
Three enzymes dealing with phosphate
Enzyme (Class) Reaction scheme / Reaction type
Kinase
substrate-OH + ATP  substrate-O-P + ADP
(Transferase)
phosphorylation = transfer of phosphoryl PO32– from ATP to substrate
Phosphatase
(Hydrolase)
substrate-O-P + H2O  substrate-OH + Pi
the hydrolysis of phosphoester bond
(glycogen)n + Pi  (glycogen)n-1 + glucose 1-P
Phosphorylase inosine + Pi  hypoxanthine + ribose 1-P
(Transferase)
phosphorolysis = the splitting of glycoside bond by
phosphate = transfer of glucosyl to inorganic phosphate
29
!
Distinguish:
Three types of lysis (decomposition of substrate)
the decomposition of substrate by water, frequent in intestine:
Hydrolysis
sucrose + H2O  glucose + fructose
(starch)n + H2O  maltose + (starch)n-2
Phosphorolysis
the cleavage of O/N-glycoside bond by phosphate:
(see previous page)
(glycogen)n + Pi  (glycogen)n-1 + glucose 1-P
the cleavage of C-C bond by sulfur atom of coenzyme A
Thiolysis
in β-oxidation of FA or ketone bodies catabolism
RCH2COCH2CO-SCoA + CoA-SH  RCH2CO-SCoA + CH3CO-SCoA
CH3COCH2CO-SCoA + CoA-SH  2 CH3CO-SCoA
30
Cofactors of enzymes
• low-molecular non-protein compounds
• many of them are derived from B-complex vitamins
• many of them are nucleotides
• transfer 2 H or e- (cooperate with oxidoreductases)
• transfer groups (cooperate with transferases)
• tightly (covalently) attached – prosthetic groups
• loosely attached – coenzymes (cosubstrates)
31
Three different components in enzyme reaction
enzyme
substrate +
cofactor

product
+
cofactoraltered
1. substrate(s)
2. cofactor
react to each other
3. enzyme catalyzes the whole process
Notes:
• one or two substrates may be involved (dehydrogenation × transamination)
• substrate can be low / high molecular (hexokinase × protein kinase)
• some reactions proceed without cofactor (hydrolysis, isomeration)
• reaction can be reversible or irreversible (dehydrogenation × decarboxylation)
32
Cofactors of oxidoreductases
Oxidized form
Reduced form
The function of cofactor
NAD+
NADH+H+
NAD+ acceptor of 2H
NADP+
NADPH+H+
NADPH+H+ donor of 2H
FAD
FADH2
FAD acceptor of 2H
Dihydrobiopterin (BH2)
tetrahydrobiopterin (BH4)
BH4 donor of 2H
Molybdopterinoxid
molybdopterinred
electron transfer
Lipoate (-S-S-)
dihydrolipoate (2 -SH)
antioxidant / transfer of acyl
Ubiquinone (Q)
ubiquinol (QH2)
transfer of 2 electrons + 2 H+
Heme-Fe3+
heme-Fe2+
transfer of 1 electron
Non-heme-S-Fe3+
non-heme-S-Fe2+
transfer of 1 electron
Glutathioneoxid (G-S-S-G)
glutathionered (GSH)
2 GSH donor of 2H
33
NAD+ is the cofactor of dehydrogenases,
derivative of nicotinamide (vitamin)
• NAD+ is oxidant – takes off 2 H from substrate
• one H adds as hydride ion
(H-)
2 H = H– + H+
into para-position of pyridinium cation of NAD+
• NAD+ + H- = NADH = equivalent of two electrons
• the second H is released as proton (H+) and
binds to enzyme molecule
34
NAD+ (nicotinamide adenine dinucleotide)
addition
of hydride aniontu
anion
adice hydridového
CONH2
anhydride
anhydrid
pyridinium
ester
N
CH2
N-glycosidic
N-glykosidová
linkage
vazba
O
O
N
N
ester
N
O P O P O CH2
OH
OH HO
adenine
adenin NH2
OH
N-glykosidová
N-glycosidic
vazba
linkage
O
difosforečná
kys.
diphosphate
N
O
ribosa
ribose
OH
OH
ribosa
ribose
35
Redox pair of cofactor
H
H
H
H
CONH2
N
!
CONH2
N
+
oxidized
form
NAD
oxidovaná forma
reduced
form NADH
redukovaná
forma
aromatic ring
aromaticity totally disturbed
aromatický kruh
tetravalent
nitrogen
kladný
náboj na N
aromaticita porušena
trivalent
nitrogen
elektroneutrální
positive charge on nitrogen
electroneutral species
high-energy compound
36
Dehydrogenation by
+
NAD
• typical substrate groups:
• primary alcohol -CH2-OH
• secondary alcohol >CH-OH
• secondary amine >CH-NH2
• double bond (C=O, C=N) is produced
37
NAD+ dehydrogenations form a double bond
Substrate
Product
primary alcohol
aldehyde
secondary alcohol
ketone
aldehyde hydrate
carboxylic acid
hemiacetal
ester
cyclic hemiacetal
lactone
hydroxy acid
oxo acid
amino acid
imino acid
compare
Med. Chem. II
Appendix 3
38
Dehydrogenation of ethanol
(alcohol dehydrogenase)
H H
H3C C O + NAD
H
H3C C
O
+
NADH+ H
H
39
Dehydrogenation of glutamate
(glutamate dehydrogenase)
H
COOH
COOH
N C H
H
CH2
HN C
+ NAD
CH2
CH2
CH2
COOH
COOH
glutamate
2-aminokyselina
+ NADH + H
2-imino glutarate
2-iminokyselina
40
NAD+-dependent enzymes are called
pyridine dehydrogenases
• Citrate cycle
isocitrate dehydrogenase
2-oxoglutarate dehydrogenase
malate dehydrogenase
• Glycolysis
glyceraldehyde 3-P dehydrogenase
lactate dehydrogenase
• Oxidation of ethanol
alcohol dehydrogenase
acetaldehyde dehydrogenase
41
Reduced cofactor NADPH+H+ is hydrogenation agent
• donor of 2 H in hydrogenations
• cofactor of reducing syntheses (FA, cholesterol)
• regeneration of glutathione (GSH) in erythrocytes
• cofactor of hydroxylation reactions:
cholesterol   bile acids
calciol   calcitriol
xenobiotic  hydroxylated xenobiotic
• general scheme of hydroxylation:
R-H + O2 + NADPH+H+  R-OH + H2O + NADP+
42
FAD is cofactor of flavin dehydrogenases,
derivative of riboflavin (vitamin B2)
• flavin adenine dinucleotide
• dehydrogenation of -CH2-CH2- group
• two H atoms are attached to two nitrogens of riboflavin
(N-1 and N-10)
• FAD + 2H  FADH2
43
FAD (flavin adenine dinucleotide)
dimethylisoalloxazine
O
H3C
N
H3C
N
NH
N
O
thevazba
sites for
2H accepting
two H atoms
CH2
CH OH
ribitol
NH2
CH OH
CH OH
CH2
N
O
OH
N
O
N
O P O P O CH2
adenine
N
O
OH
difosfát
diphosphate
OH
OH
ribosa
ribose
44
!
Redox pair of cofactor
O
HN
O
N
O
10
N
CH3
N
CH3
1
HN
O
N
H
N
CH3
N
CH3
H
oxidovaná forma
oxidized
form FAD
redukovaná
reduced form
FADH2forma
aromatic system
aromaticity partially disturbed
electroneutral species
electroneutral species
high-energy compound
45
Dehydrogenation of succinate to fumarate
(flavin dehydrogenase)
COOH
CH2
CH2
H
+ FAD
HOOC
C
C
COOH
+
FADH2
H
COOH
46
Tetrahydrobiopterin (BH4) is a cofactor of hydroxylations
O
H
N
HN
H2N
OH
N
CH3 1,2-dihydroxypropyl
OH
N
H
tetrahydropteridin
O
H2N
• donor of 2H
N
HN
N
guanine
• made in the body from GTP
• oxidized to dihydrobiopterin (BH2)
N
H
47
Redox pair of cofactor
O
HN
H
N
H
N
H
N
N
H
OH
O
CH3
OH
tetrahydrobiopterin
(BH 4)
N
HN
- 2H
HN
OH
N
N
H
CH3
OH
dihydrobiopterin
(BH 2)
48
Hydroxylation of phenylalanine
COOH
H2N CH
COOH
+
O O + BH4
H2N CH
CH2
CH2
H
OH
fenylalanin
phenylalanine
+
H2O + BH2
tyrosin
tyrosine
49
Coenzyme Q (ubiquinone)
• derivative of 1,4-benzoquinone
O
O
• cyclic diketone, not aromatic
CH3
H3C
CH3
O
CH3
O
CH3
CH3
CH3
• component of respiratory chain
• gradually accepts electron and proton (2x)
• reduced to semiubiquinone and ubiquinol
50
Reversible reduction of ubiquinone
Q  •QH  QH2
O
H3CO
CH3
H3CO
OH
OH
e
e
+ H
+ H
R
O
ubiquinone
ubichinon
(non-aromatic cycl. diketone)
OH
O
semiubiquinone
semiubichinon
ubichinol
ubiquinol
(aromatic ring + radical)
(diphenol)
electron (e-) and proton (H+) have different origin:
electron comes from reduced cofactors, H+ from matrix of mitochondria
R = long polyisoprenoid chain  lipophilic character
51
Heme of various cytochromes
• transfers just 1 electron
• cytochromes are hemoproteins
• components of respiratory chain or other heme enzymes (cyt P-450)
• reversible redox reaction: Fe2+  Fe3+
N
N
N
Fe 3+
Fe 2+
N
N
N
N
N
52
Non-heme iron (Fe2S2 cluster) transfers electron in R.CH.
Cys
S
S
Fe
3+
Fe
Cys
Cys
S
S
+ e
3+
S
Cys
oxidovaný
stav
oxidized state
S
Fe
- e
S
Cys
S
S
Cys
Cys
3+
S
Fe
2+
S
S
Cys
redukovaný
stav
reduced state
just one iron cation changes oxidation number
53
Molybdopterin (formula in Seminars)
Xanthine oxidase catalyzes
the oxygenation of purine bases (catabolism)
N
N
N
N
OH
OH
OH
HO
N
H
hypoxanthine

N
N
N
HO
N
H
xanthine

N
N
N
H
OH
uric acid
side product: H2O2
54
Molybdopterin
Sulfite oxidase:
sulfate is catabolite from cysteine
cysteine
HSO3- + H2O  SO42- + 3 H+ + 2 eplasma
urine
acidify
plasma
urine
reduce Mo
55
Redox pair lipoate/dihydrolipoate is antioxidant system.
It is also involved in the acyl transfer (see later)
CONH
S
S
2H
CONH
H
Enzym
oxidized form – lipoate
lipoát
(cyclic disulfide 1,2-dithiolane)
- 2H
S
Lys
S
H
Lys
Enzym
one S atom transfers acyl in oxidative
decarboxylation of pyruvate / 2-oxoglutarate
reduced
form - dihydrolipoate
dihydrolipoát
56
Glutathione (GSH)
• tripeptide
• γ-glutamyl-cysteinyl-glycine
• cofactor of glutathione peroxidase (contains selenocysteine)
• reduces H2O2 to water
• 2 G-SH + H-O-O-H  G-S-S-G + 2 H2O
Remember:
The -SH compounds have generally reducing properties.
57
Dehydrogenation of two GSH molecules
NH2
H
N
HOOC
2
NH2
H
HOOC
N
O
O
CH2
- 2H
N
N
O
COOH
H
S
COOH
S
O
CH2
SH
H
H
HOOC
CH2
O
COOH
N
N
O
H
NH2
58
Vitamins and cofactors of transferases
Vitamin
Cofactor
Transferred group
Pyridoxin
pyridoxal phosphate
-NH2 (transamination)
(Made in body)
ATP
-PO32- (phosphoryl)
(Made in body)
PAPS
-SO32-
Biotin
carboxybiotin
CO2
Pantothenic acid
CoA-SH
acyl
(Made in body)
dihydrolipoate
acyl
(Methionine)
SAM
-CH3
Folate
tetrahydrofolate
C1 groups
Cyanocobalamin
methylcobalamin
-CH3
Thiamin
thiamin diphosphate
residue of oxo acid
59
Pyridoxal phosphate is the cofactor
of transamination and decarboxylation of AA
R CH COOH
R CH COOH
NH2
H
C
HO
O
N
CH
CH2O P
N
- H2O
O
OH
H 3C
transamination
decarboxylation
OH
N
aldimine
(Schiff base)
60
ATP is the cofactor of kinases (phosphorylation agent)
NH2
anhydride
ester
O
O
N
O
N
O P O P O P O
O
O
O
O
OH
N
N
N-glycoside
bond
OH
61
Phosphorylation of substrate
O
OH
O
O
Ade
O P O P O P O Rib
+
O
O
O
ATP (4-)
substrát
substrate
CAUTION: creatine kinase (CK)
phosphorylation on nitrogen (the bond N-P)
kinase
kinasa
O
O
O P O
O
O
Ade
+ O P O P O Rib + H
O
O
fosforylovaný substrát
(2-)
phosphorylated
substrate
ADP (3-)
62
PAPS is sulfation agent
•
•
NH2
3’-phosfoadenosine-5’-phosphosulfate
mixed anhydride of
H2SO4 and H3PO4
•
esterification of hydroxyl groups by
sulfuric acid = sulfation
•
sulfated sphingoglycolipids
•
sulfated glycosaminoglycans (heparin,
chondroitin sulfate, keratan sulfate)
N
N
N
N
O
O
O S
O P
O
O
O CH2
O
O
O P
OH
O
O
63
Carboxybiotin
• cofactor of carboxylation reactions
• carboxylation of biotin needs ATP
ATP
CO2
O
HN
ADP + P
O
O
C
NH
N
NH
HO
S
biotin
biotin
COOH
S
COOH
karboxybiotin
carboxybiotin
64
O
O
Carboxybiotin is the cofactor
of carboxylation reactions
C
N
NH
HO
COOH
S
Biotin H
Biotin COOH
pyruvate carboxylase
pyruvátkarboxylasa
+
H3C C COOH
O
pyruvát
pyruvate
HOOC CH2
C COOH
O
oxalacetát
oxaloacetate
65
Distinguish: Decarboxylation vs. Carboxylation
!
Cofactor
Decarboxylation (does not require energy)
Thiamin-diP
pyruvate  acetyl-CoA + CO2
2-oxoglutarate  succinyl-CoA + CO2
Pyridoxal-P
amino acid  amine + CO2
None
acetoacetate  acetone + CO2 (non-enzymatic, spontaneous)
Cofactor
Carboxylation (requires energy)
Biotin
pyruvate + CO2 + ATP  oxaloacetate
acetyl-CoA + CO2 + ATP  malonyl-CoA
propionyl-CoA + CO2 + ATP  methylmalonyl-CoA  succinyl-CoA
carboxylations (ATP) in the catabolism of Val, Leu, Ile
Phylloquinone
(vitamin K)
protein-glutamate + O2 + vit Kred + CO2  protein-γ-carboxyglutamate
posttranslational carboxylation of glutamate  hemostasis
None
Hb-NH2 + CO2  Hb-NH-COOH (unstable Hb-carbamate, spontaneous)
66
Coenzyme A (CoA-SH)
• transfers acyl
• attached to sulfur atom
O
R C
• thioester bond
• acyl-CoA is activated acyl
• e.g. acetyl-CoA
67
Coenzyme A
acyl
NH2
N
N
CH3
O
N
N
O
C CH C CH2 O P~O P O CH2
O
C CH2 CH 2 HN
HS CH2 CH 2 HN
cysteamine
O
β-Alanine
HO
CH3
O
O
O
Pantoic acid
pantothenic acid
O
OH
O P O
O
3´-PhosphoADP 68
Lipoate (lipoamide)
part of the 2-oxo acid dehydrogenase complex (see the following lectures)
it is oxidant of a group carried by thiamine diphosphate (TDP),
binds the resulting acyl as thioester and transfers the acyl to coenzyme A:
CO–NH–Lys–Enzyme
Lipoamide
(oxidized form)
S6-Acyldihydrolipoamide
(reduced form)
Dihydrolipoamide
(reduced form)
S
S
R1-CH–TDP
OH
H–TDP
CO–NH–Lys–Enzyme
S
H
S
R-CO
–2H
CoA-SH
R-CO–S-CoA
CO–NH–Lys–Enzyme
S
H
S
H
69
S-Adenosylmethionine (SAM)
• „active methyl“, trivalent sulfur  sulfonium cation
• cofactor of methylation reactions:
ethanolamine → choline (3× methylation)
guanidine acetate → creatine
noradrenaline → adrenaline ..... and many others
• side product is homocysteine
• remethylation of homocysteine needs methyl-FH4 + B12 cofactor
(see Seminars)
70
S-Adenosylmethionine (SAM)
NH2
N
N
CH3
N
N
S
HOOC CH CH2CH2
NH2
O
OH
OH
71
Folic acid is vitamin.
In the body, it is hydrogenated to 5,6,7,8-tetrahydrofolate.
OH
N
H2N
O
5
N 6 CH2NH
N
N
pteridin 8
pteridine
7
COOH
glutamová
kys.
C NH CH glutamic
acid
amid
amide
CH2
p-aminobenzoová acid
kys.
p-aminobenzoic
CH2
COOH
Tetrahydrofolate (FH4) is cofactor for the transfer of C1 groups
přenos jednouhlíkatých skupin
transfer of 1C groups
OH
N
H2N
H
N
5
N
N
H
O
CH2NH
10
C
COOH
NH CHCH2CH2COOH
72
C1 Groups transferred by FH4
Oxidation
number of C
compare scheme
Seminars, p. 26
Formula
Name
Metabolic Origin / Comment
-III
-CH3
methyl
reduction of methylene-FH4 (from serine, glycine)
methyl-FH4 cooperates with B12 cofactor in methylation
-II
-CH2-
methylene
catabolism of serine, glycine
used in synthesis of dTMP  DNA
-I
-CH=
methenyl
deamination of formimino-FH4 (from histidine)
used in synthesis of purine bases
+I
-CH=O
formyl
catabolism of tryptophan  formiate  formyl
used in synthesis of purine bases
+I
-CH=NH
formimino
catabolism of histidine
73
B12 vitamin is cyano or hydroxocobalamin
R = CN or OH
corrin cycle
hydroxocobalamin is used in the treatment
of cyanide poisoning, it binds cyanide ions
to nontoxic cyanocobalamin
74
B12 cofactor is methyl or deoxyadenosylcobalamin,
it is needed for two reactions in the body
FH4 / B12
1.
homocysteine  methionine
methylation of homocysteine (regeneration of methionine)
2.
B12
homocysteine   propionyl-CoA   succinyl-CoA
75
Compare: Four different cofactors of methylations
Cofactor
SAM
Origin of methyl
methionine
Examples of methylation reactions
ethanolamine  choline
guanidine acetate  creatine
noradrenaline  adrenaline
methylation of DNA (regulation of gene expression)
methylation of bases in tRNA / mRNA (guanine-N7 = cap)
inactivation of catecholamines (COMT):
• dopamine  methoxytyramine
• noradrenaline  normetanephrine
• adrenaline  metanephrine
methylation of xenobiotics (II. phase - conjugation)
methyl-FH4
methylene-FH4
homocysteine  methionine
methyl-B12
methyl-FH4
methylene-FH4
serine, glycine
dUMP  dTMP
dUMP + methylene-H4F  dTMP + H2F (thymidylate synthase)
SAM = S-adenosylmethionine, FH4 = tetrahydrofolate, COMT = catechol O-methyltransferase
76
Thiamin is vitamin B1
Thiamin diphosphate (TDP) is cofactor
CH3
N
H3C
N
N
NH2
CH2CH2OH
S
Oxidative decarboxylation of some 2-oxo acids
• pyruvate  acetyl-CoA
• 2-oxoglutarate  succinyl-CoA (citrate cycle)
• 2-oxo acids in the catabolism of branched amino acids (Val, Leu, Ile)
TDP
TDP
TDP
H
R C COOH
R C COOH
OH
- CO2
R CH
transfer to
dihydrolipoate and CoA
OH
O
Transketolase reactions in pentose cycle
• ribose-5-P + xylulose-5-P  glyceraldehyde-3-P + sedoheptulose-7-P
• xylulose-5-P + erythrose-4-P  fructose-6-P + glyceraldehyde-3-P
77
Thiamin diphosphate (TDP) is cofactor
in the oxidative decarboxylation of pyruvate
CH3
N
H3C
O
N
N
NH2
CH2CH2
S
O
O P O P O
O
O
attachment of pyruvate and its decarboxylation
TDP
glucose  pyruvate  acetyl-CoA
CAC
78
In human body, a number of non-enzymatic reactions proceeds
•
decarboxylation of acetoacetate  acetone
•
catabolism of creatine  creatinine (dehydration + cyclization)
•
glycation / carbamylation / nitrosylation / nitration of proteins
•
the reactions of reactive oxygen species (e.g. lipoperoxidation)
•
spontaneous oxidation of hemoproteins (hemoglobin  methemoglobin)
•
spontaneous oxidation of urobilinogens to urobilins (large intestine)
•
condensation of amines with carbonyl compounds to heterocyclic derivatives
dopamine + pyruvate  salsolinol (neurotoxin ?)
tryptamine + pyruvate  harmane
dopamine + dihydroxyphenylacetaldehyde  tetrahydropapaveroline
•
binding ligands to proteins:
bilirubin + albumin  bilirubin-albumin complex
CO + hemoglobin  carbonylhemoglobin
•
the interactions of macromolecules:
antigen + antibody  immuno complex
79