Enzymes I

General features, cofactors

 Department of Biochemistry, FM MU, 2011 (J.D.)

1
Literature for Biochemistry I

• Lecture files on is.muni.cz.

• Tomandl J., Táborská E.: Biochemistry I – Seminars. MU, 2008.

• Harvey R.A., Ferrier D.R.: Biochemistry. 5th ed.,

Lippincott Williams & Wilkins, 2011.

2
General features of enzymes
CAUTION: peptidyltransferase is ribozyme

rRNA
(AK)n-tRNA1 + AK-tRNA2 → tRNA1 + (AK)n+1-tRNA2
• biocatalysts
• 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
• 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
Enzymes can be regulated
(see the lecture Enzymes II)

Activity of enzyme Quantity of enzyme

• activators • regulation of proteosynthesis
• inhibitors and proteolysis of enzyme

• covalent modification • some hormones act as

(phosphorylation) inducers × repressors

6
Dual specifity of enzymes towards:

Reaction Substrate
catalyze just work with one substrate
(or group of similar substrates)
one type of reaction
often stereospecific

7
 Enzymes are stereospecific catalysts
there are two types of stereospecific conversions:

• non-chiral substrate → chiral product(one enantiomer)

pyruvate → L-lactate

fumarate → L-malate

• 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

8
 1. non-chiral substrate → chiral product
Hydrogenation of pyruvate
When pyruvate is hydrogenated without enzyme (in vitro),
the reaction product is the racemic mixture of D-lactate and L-lactate:
H
COOH COOH
O CH3
C C HO C H + H C OH
HO O
CH3 CH3

H L-laktát
L-Lactate + D-laktát
D-Lactate

In the same reaction catalyzed by lactate dehydrogenase (in the presence

of NADH+H+), pyruvate is reduced stereospecifically to L-lactate only:
H
COOH
O CH3
C C HO C H
HO O
CH3
Enzyme L-Lactate
9
 1. non-chiral substrate → chiral product

Non-enzymatic hydration of fumarate

COOH addition from

adice z levé
H H COOH H addition
adice from
z pravé COOH
one side
strany strany side
another
HO C H O C O H C OH
CH2COOH H C H CH2COOH

L-malát
L-malate HOOC H
D-malát
D-malate

fumarate

in vitro reaction proceeds to racemic D,L-malate

10
 1. non-chiral substrate → chiral product

Enzymatic hydration of fumarate (citrate cycle)

H H reagent
O

H C C COOH
HOOC H Substrát
substrate

Enzym
enzyme

in vivo just one enantiomer (L-malate) is produced

11
 1. non-chiral substrate → chiral product

Hydrogenation of D-fructose in vitro gives two epimers

reaction site is planar

CH2OH CH2OH CH2OH

C O H C OH HO C H
HO C H 2H HO C H HO C H
+
H C OH H C OH H C OH
H C OH H C OH H C OH
CH2OH CH2OH CH2OH

D-fructose D-glucitol D-mannitol

in vivo: enzymatic reaction gives just one product (D-glucitol)

12
 2. chiral substrate → product

Enzymes or receptors recognize only one enantiomer

If the reactant of an enzymatic reaction is a chiral compound,

only one of the two enantiomers is recognized as the specific
substrate.
Chiral substrates/signal molecules are bound to the stereospecific
enzymes/receptors at three sites:
R R see also
MCH II, p. 13

X
x

proper enantiomer not-fitting enantiomer

13
 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/ 14
 Examples
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

15
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 (reverse reaction: synthases)

5. Isomerases A-B-C  A-C-B

6. Ligases (synthetases) A + B + ATP → A-B + ADP + Pi

16
1 Oxidoreductases
catalyze the oxidation or reduction of substrate

subclasses:
• dehydrogenases catalyze transfers of two hydrogen 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 breakdown of peroxides

Example: lactate + NAD+  pyruvate + NADH + H+

Recommended name: lactate dehydrogenase
Systematic name: (S)-lactate:NAD+ oxidoreductase
17
 2 Transferases
catalyze the transfer of a group from one to another substrate

subclasses:

• aminotransferases, methyltransferases, glucosyltransferases

• phosphomutases – the transfer of the group PO32– within molecule

• kinases phosphorylate substrate by the transfer
of phosphoryl group PO32– from ATP (e.g. hexokinases, proteinkinases)

Example: glucose + ATP → glucose 6-P + ADP

Recommended name: glucokinase
Systematic name: ATP:D-glucose phosphotransferase
18
Example: Phosphorylation of glucose

O
O
P
HO O
O
O OH O OH
glucokinase
glukokinasa
OH + ATP OH + ADP

OH OH
OH OH

glucose
glukosa glucose 6-phosphate
glukosa-6-fosfát

19
 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 and peptidases (pepsin, trypsin, cathepsins,
dipeptidases, carboxypeptidases, aminopeptidases)
• amidases (glutaminase, asparaginase)
• ATPases (split anhydride bonds of ATP)

Example: glucose 6-P + H2O → glucose + Pi

Recommended name of the enzyme: glucose 6-phosphatase
Systematic name: glucose 6-phosphate phosphohydrolase
20
 Example: Glucose 6-phosphatase

O
O
P
O HO
O
O OH O OH O
O
OH + H2O OH + P
OH
OH OH O

OH OH

glukosa-6-fosfát
Glc 6-P Glc
glukosa

21
 Compare two antagonistic enzymes

ATP ADP
kinase

O
Enzym Serin OH Enzym Serin O P O
O

O
phosphatase
H2O
O P OH
O
22
Glutaminase is amidase which catalyzes
the deamidation of glutamine

COOH COOH
H2N CH H2N CH
CH2 H2O CH2
+ NH3
CH2 glutaminase CH2
C C
O NH2 O OH

glutamin
glutamine glutamát
glutamate
23
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)

24
 Examples of lysosomal hydrolases
Hydrolase Bond hydrolyzed
Glucosidase glycoside
Galactosidase glycoside
Hyaluronidase glycoside
Arylsulfatase sulfoester
Lysozyme glycoside
Cathepsin peptide
Collagenase peptide
Elastase peptide
Ribonuclease phosphodiester
Lipase ester
Phosphatase phosphoester
Ceramidase amide
25
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

26
 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 (carbonate dehydratase: CO2 + H2O  H2CO3)

Example: fumarate + H2O  L-malate

Recommended name: fumarate hydratase
Systematic name: (S)-malate hydro-lyase (fumarate-forming)
27
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
28
 6 Ligases
catalyze 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)

29
Three enzymes have something to do 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 substrate-O-P + H2O → substrate-OH + Pi

(Hydrolase) 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
30
 !
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

the cleavage of O/N-glycoside bond by phosphate:

Phosphorolysis
(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

31
 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)

32
 Three different components in enzyme reaction

enzyme

substrate + cofactor  product + cofactoraltered

1. substrate(s)
react to each other
2. cofactor
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) 33
 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
34
 NAD+ is the cofactor of dehydrogenases
• 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

35
 NAD+ (nicotinamide adenine dinucleotide)

addition of hydrideaniontu
adice hydridového anion

CONH2 adenin NH2

adenine
pyridinium anhydride
anhydrid
N
N
N ester O O ester

CH2 O P O P O CH2 N N
N-glycosidic
N-glykosidová
linkage
vazba OH OH
N-glycosidic
N-glykosidová
OH HO difosforečná kys. O vazba
linkage
diphosphate
O
ribose
ribosa
OH OH
ribosa
ribose
36
 !
Redox pair of cofactor

H H
H H
CONH2 CONH2

N N

oxidized form NAD +

oxidovaná forma reduced form NADH
redukovaná forma
aromatic ring aromaticity totally disturbed
aromatický kruh aromaticita porušena
tetravalent nitrogen
kladný náboj na N trivalent nitrogen
elektroneutrální
positive charge on nitrogen electroneutral species
high-energy compound 37
 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

38
NAD+ dehydrogenations form a double bond

Substrate Product

primary alcohol aldehyde

secondary alcohol ketone
aldehyde hydrate carboxylic acid
compare
hemiacetal ester Med. Chem. II
cyclic hemiacetal lactone Appendix 3
hydroxy acid
oxo acid
amino acid
imino acid
39
 Dehydrogenation of ethanol
(alcohol dehydrogenase)

H H
O
H3C C O + NAD H3C C + NADH+ H
H H

40
 Dehydrogenation of glutamate
(glutamate dehydrogenase)

H COOH COOH
N C H HN C
H CH2 + NAD CH2 + NADH + H
CH2 CH2
COOH COOH

2-aminokyselina
glutamate 2-iminokyselina
2-imino glutarate

41
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
42
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+

43
 FAD is cofactor of flavin dehydrogenases

• 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

44
 FAD (flavin adenine dinucleotide)

dimethylisoalloxazine
O
H3C N
NH thevazba
sites for
2H accepting
two H atoms
H3C N N O
CH2
CH OH NH2
ribitol CH OH adenine
N
N
CH OH O O
CH2 O P O P O CH2 N N
OH OH O

difosfát
diphosphate
ribosa
ribose
OH OH
45
 O 10
N
Redox pair of cofactor

CH3
O H
N CH3
!
HN HN

O N N CH3 O N N CH3
1
H
oxidovaná forma redukovaná
oxidized form FAD reduced form FADH2forma

aromatic system aromaticity partially disturbed

electroneutral species electroneutral species
high-energy compound
46
 Dehydrogenation of succinate to fumarate
(flavin dehydrogenase)

COOH H COOH
C
CH2 + FADH2
+ FAD C
CH2 HOOC H
COOH

47
Tetrahydrobiopterin (BH4) is a cofactor of hydroxylations

O OH
H
N CH3 1,2-dihydroxypropyl
HN
OH
H2N N N
H
tetrahydropteridin • made in the body from GTP
O
• donor of 2H
N
HN • oxidized to dihydrobiopterin (BH2)

H2N N N
H
guanine 48
 Redox pair of cofactor

O OH O OH
H
N CH3 N CH3
HN HN
H OH - 2H OH
N N N HN N N
H H
H

tetrahydrobiopterin dihydrobiopterin
(BH4) (BH2)

49
 Hydroxylation of phenylalanine

COOH COOH
H2N CH + O O + BH4 H2N CH + H2O + BH2
CH2 CH2

H OH
fenylalanin
phenylalanine tyrosin
tyrosine

50
 Coenzyme Q (ubiquinone)
• derivative of 1,4-benzoquinone
O

• cyclic diketone, not aromatic H3C

O CH3

CH3
O

• component of respiratory chain CH3 O CH3 CH3 CH3

• gradually accepts electron and proton (2x)

• reduced to semiubiquinone and ubiquinol

51
 Reversible reduction of ubiquinone
Q  •QH  QH2
O OH OH
H3CO CH3
e + H e + H

H3CO R
O OH
O

ubiquinone
ubichinon semiubiquinone
semiubichinon ubichinol
ubiquinol
(non-aromatic diketone) (aromatic ring + radical) (diphenol)

electron (e-) and proton (H+) have different origin:

electron comes from red. cofactors (= nutrients), H+ from matrix of mitochondrion

R = long polyisoprenoid chain ⇒ lipophilic character 52

 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 N
Fe 2+ Fe 3+

N N N N

53
 Non-heme iron (Fe2S2 cluster) transfers electron in R.CH.

Cys Cys Cys Cys

S S S S S S
+ e
3+ 3+ 3+ 2+
Fe Fe Fe Fe
- e
S S S S S S
Cys Cys Cys Cys

oxidized state
oxidovaný stav redukovaný stav
reduced state

just one iron cation changes oxidation number

54
Molybdopterin (formula in Seminars)
Xanthine oxidase catalyzes
the oxygenation of purine bases
(catabolism)
OH OH OH

N N N N N N

N N HO N N HO N N OH
H H H

hypoxanthine → xanthine → uric acid

side product: H2O2

55
Molybdopterin
Sulfite oxidase:
sulfate is catabolite from cysteine

cysteine

HSO3- + H2O → SO42- + 3 H+ + 2 e-

plasma
urine acidify
plasma reduce Mo
urine
56
(see Seminars, p. 46)
Redox pair lipoate/dihydrolipoate is antioxidant system.
It is also involved in the acyl transfer (see later)

CONH Lys Enzym

oxidized form – lipoate
S S lipoát
(cyclic disulfide 1,2-dithiolane)

- 2H 2H

CONH Lys Enzym

one S atom transfers acyl in oxidative
S S decarboxylation of pyruvate / 2-oxoglutarate
H H
dihydrolipoát
reduced form - dihydrolipoate 57
 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.

58
 Dehydrogenation of two GSH molecules

NH2 H O

N
HOOC N COOH

O CH2 H

2
NH2 H O

N
- 2H S
HOOC N COOH
S
O CH2 H
H CH2 O
SH
HOOC N COOH
N

O H NH2

59
 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
60
Pyridoxal phosphate is the cofactor
of transamination and decarboxylation of
AA

R CH COOH
R CH COOH
NH 2 transamination
N
H O CH
C O - H2O
HO CH 2O P OH decarboxylation

OH
H 3C N N
aldimine
(Schiff base)

61
Two phases of transamination
blue colour indicates the pathway of nitrogen

amino acid
aminokyselina
R CH COOH R CH COOH R C COOH R C COOH
NH2 N N H2O O oxokyselina
oxo acid

H O - H2O CH CH2 CH2NH2

C

Schiff base
pyridoxal pyridoxamin

HOOC C CH2CH2COOH HOOC CH CH2CH2COOH

O 2-oxoglutarát
2-oxoglutarate NH2 glutamát
glutamate
H2O
- H2O H O
CH2NH2 C

62
pyridoxamin pyridoxal
ATP is the cofactor of kinases (phosphorylation agent)

anhydride NH2
ester
N
N
O O O
N N
O P O P O P O
O O O O N-glycoside
bond

OH OH
63
Phosphorylation of substrate
O O O Ade

OH + O P O P O P O Rib
O O O

substrát
substrate ATP (4-)

CAUTION: creatine kinase (CK)

phosphorylation on nitrogen (the bond N-P) kinasa
kinase

O O O Ade

O P O + O P O P O Rib + H
O O O
64
fosforylovaný substrát
phosphorylated (2-)
substrate ADP (3-)
 PAPS is sulfation agent
• 3’-phosfoadenosine-5’-phosphosulfate NH2

• mixed anhydride of N
N
H2SO4 and H3PO4 N
N
• esterification of hydroxyl groups by O O

sulfuric acid = sulfation

O S O P O CH2
O
O O
• sulfated sphingoglycolipids

• sulfated glycosaminoglycans (heparin,

O OH
chondroitin sulfate, keratan sulfate)
O P O
O
65
 Carboxybiotin
• cofactor of carboxylation reactions
• carboxylation of biotin needs ATP

CO2 ATP
O O
ADP + P
O
HN NH C N NH
HO

COOH S COOH
S

biotin
biotin carboxybiotin
karboxybiotin

66
 O

Carboxybiotin is the cofactor O

C N NH

of carboxylation reactions HO

S COOH

Biotin COOH Biotin H

pyruvate carboxylase
pyruvátkarboxylasa
+

H3C C COOH HOOC CH2 C COOH

O O

pyruvát
pyruvate oxalacetát
oxaloacetate 67
 !
Distinguish: Decarboxylation vs. Carboxylation

Cofactor Decarboxylation (does not require energy)

pyruvate → acetyl-CoA + CO2
Thiamin-diP
2-oxoglutarate → succinyl-CoA + CO2

Pyridoxal-P amino acid → amine + CO2

None acetoacetate → acetone + CO2 (non-enzymatic, spontaneous)

Cofactor Carboxylation (requires energy)

pyruvate + CO2 + ATP → oxaloacetate
acetyl-CoA + CO2 + ATP → malonyl-CoA
Biotin
propionyl-CoA + CO2 + ATP → methylmalonyl-CoA → succinyl-CoA
carboxylations (ATP) in the catabolism of Val, Leu, Ile
Phylloquinone protein-glutamate + O2 + vit Kred + CO2 → protein-γ-carboxyglutamate
(vitamin K) posttranslational carboxylation of glutamate → hemostasis
None Hb-NH2 + CO2 → Hb-NH-COOH (unstable Hb-carbamate, spontaneous)
68
 Coenzyme A (CoA-SH)

O
• transfers acyl
• attached to sulfur atom
R C
• thioester bond
• acyl-CoA is activated acyl
• e.g. acetyl-CoA

69
 Coenzyme A

acyl NH2

N
N

CH3 O O N N
O
O C CH C CH 2 O P~O P O CH 2
C CH2 CH 2 HN CH3 O O
HO O
HS CH2 CH 2 HN

Cysteamine β-Alanine Pantoic acid

Pantothenic acid O OH
O P O
O
3´-PhosphoADP 70
 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 R1-CH–TDP
(oxidized form) S S
OH
H–TDP
–2H
CO–NH–Lys–Enzyme
S6-Acyldihydrolipoamide
(reduced form) S S CoA-SH
H
R-CO R-CO–S-CoA

Dihydrolipoamide CO–NH–Lys–Enzyme
(reduced form) S S
H H

71
 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, p. 27 and 45)

72
S-Adenosylmethionine (SAM)
NH2
N
N

N N
CH 3
S
HOOC CH CH2CH2
O
NH2

OH OH
73
Folic acid is vitamin.
In the body, it is hydrogenated to 5,6,7,8-tetrahydrofolate.
OH 5 O COOH
N 6 CH2NH glutamová
C NH CH glutamic kys.
acid
N amid
amide CH2
7 p-aminobenzoová acid
kys.
H2N N N p-aminobenzoic CH2
pteridin 8 COOH
pteridine

Tetrahydrofolate (FH4) is cofactor for the transfer of C1 groups

přenos jednouhlíkatých skupin
transfer of 1C groups

OH O COOH
H
N CH2NH C NH CHCH2CH2COOH
N 5 10

H2N N N 74
H
 compare scheme
C1 Groups transferred by FH4
Seminars, p. 26

Oxidation
number of C
Formula Name Metabolic Origin / Comment

reduction of methylene-FH4 (from serine, glycine)

-III -CH3 methyl methyl-FH4 cooperates with B12 cofactor
in methylation

catabolism of serine, glycine

-II -CH2- methylene
used in synthesis of dTMP → DNA

deamination of formimino-FH4 (from histidine)

-I -CH= methenyl
synthesis of purine bases

catabolism of tryptophan → formiate → formyl

+I -CH=O formyl
synthesis of purine bases

+I -CH=NH formimino catabolism of histidine

75
B12 vitamin is cyano or hydroxocobalamin

R = CN or OH
corrin cycle

hydroxocobalamin is used in the treatment

of cyanide poisoning - binds cyanide ions
to non-toxic cyanocobalamin 76
B12 cofactor is methyl or deoxyadenosylcobalamin,

it is needed for two reactions in the body

FH4 / B12
• homocysteine → methionine
methylation of homocysteine (regeneration of methionine)

B12
• homocysteine → → propionyl-CoA → succinyl-CoA

you can see both reactions in Seminars, p. 45

77
 !
Compare: Four different cofactors of methylations
Cofactor Methyl origin Examples of methylation reactions

ethanolamine → choline
guanidineacetate → creatine
noradrenaline → adrenaline
the methylation of DNA (regulation of gene expression)
SAM methionine methylation of bases in tRNA / mRNA (guanine-N7 = cap)
the inactivation of catecholamines (COMT):
• dopamine → methoxytyramine
• noradrenaline → normetanephrine
• adrenaline → metanephrine
the methylation of xenobiotics (II. phase - conjugation)

methyl-FH4 methylene-FH4 homocysteine → methionine O O

H CH3
HN HN
methylene-FH4 serine, glycine dUMP → dTMP -CH2-

O N O N
H H
methyl-B12 methyl-FH4 homocysteine → methionine

SAM = S-adenosylmethionine, FH4 = tetrahydrofolate, COMT = catechol O-methyltransferase 78

 CH3
Thiamin is vitamin B1 N N
CH2CH2OH
Thiamin diphosphate (TDP) is cofactor H3C N NH2 S

Oxidative decarboxylation of some 2-oxo acids

• pyruvate → acetyl-CoA
• 2-oxoglutarate → succinyl-CoA (citrate cycle)
• 2-oxo acids in the catabolism of Val, Leu, Ile

TDP
TDP TDP transfer to
H
R C COOH R CH dihydrolipoate and CoA
- CO2 OH
R C COOH OH
O

Transketolase reactions in pentose cycle

• ribose-5-P + xylulose-5-P  glyceraldehyd-3-P + sedoheptulose-7-P
• xylulose-5-P + erythrose-4-P  fructose-6-P + glyceraldehyd-3-P
79
Thiamin diphosphate (TDP) is the cofactor
of the oxidative decarboxylation of pyruvate

CH3
O O
N N
CH2CH2 O P O P O
H3C N NH2 S O O

attachment of pyruvate and its decarboxylation

TDP
glucose → pyruvate → acetyl-CoA

CAC 80
In human body, a number of non-enzymatic reactions proceeds
• decarboxylation of acetoacetate → acetone
• glycation / carbamylation / nitrosylation / nitration of proteins
• the reactions of ROS (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
tryptamine + pyruvate → harmane neurotoxins
dopamine + dihydroxyphenylacetaldehyde → tetrahydropapaveroline
• binding ligands to proteins:
bilirubin + albumin → bilirubin-albumin complex
CO + hemoglobin → carbonylhemoglobin
• the interactions of macromolecules:
antigen + antibody → immuno complex
81