Enzyme cofactors

1
Enzyme cofactors

• Jana Novotna

2
Content of lectures

• Introduction into cofactors
• explanation of the differences between cofactors
• Types of cofactors (enzymatic reactions in large
  multienzyme complexes involving several
  cofactors)
• cofactors derived from vitamins (part of large
  complexes such as multienzyme pyruvate
  dehydrogenase)
• cofactors of oxidoreductases (NAD, NADP, FAD,
  FMN)
• cofactors of transferases (ATP, pyridoxal
  phosphate, tetrahydrofolate etc.)
• The importance of vitamins as cofactors (vitamin
  C, A, E, K)
• What are ferredoxins, methalloporphyrins.

3
Cofactors importance

• Cofactor non-protein, low molecular compound of
  enzyme
• Cofactor - a co-catalyst required for enzyme
  activity, "helper molecules" that assist in
  biochemical transformations.
• Apoenzyme - enzyme lacking cofactor (inactive)
• Holoenzyme - enzyme with cofactors (active)
• Classification
• loosely-bound cofactors termed coenzymes
• tightly-bound cofactors termed prosthetic groups
• Some enzymes or enzyme complexes require several
  cofactors (pyruvate dehydrogenase multienzyme
  complex)

4
Difference between the coenzyme and prosthetic
group

• Coenzyme act as second substrate ? the enzyme
  reaction is completed ? coenzyme is transferred
  to another apoenzyme ? it regenerates when reacts
  with another substrate (coupled reaction).
• Prosthetic group covalently bound to the enzyme
  regenerates in succession with two different
  substrates.

5

• Organic cofactors
• vitamins or vitamin derivatives (water-soluble
  all B vitamins, vitamin C, lipid-soluble K, A,
  E
• heterocycles with the rest of the molecule of
  phosphoric acid
• many cofactors contain the nucleotide (AMP) as
  part of their structure
• Inorganic cofactors
• metal ions Mg2, Cu, Mn2, Zn2, Se, iron-sulfur
  clusters

6
(No Transcript)
7
Cofactor Vitamin Enzyme reaction (example)
Thiamine diphosphate Thiamine, B1 Oxidative decarboxylation
FAD, FMN Riboflavin, B2 Redox reactions (transfer of H)
NAD, NADP Niacin, B3 Redox reactions (transfer of H)
Coenzyme A Pantothenic acid, B5 Metabolism of FA (transfer of acyl groups )
Pyridoxalphosphate Pyridoxine, B6 Transamination, decarboxylation of AA
Carboxybiotin Biotin, H, B7 Carboxylation (CO2)
Tetrahydrolic acid Folic acid, B9 Transfer of C1
Ascorbic acid Vitamin C Hydroxylation reaction (collagen)
cis-retinal opsin Retinol, A Electron transport , antioxidant
? Tocoferol, E Redox reaction, antioxidant
Menaquinone Vitamin K Transfer of carbonyl group and electrons
Lipoamide Lipoic acid Transfer of electrons and acyl group
Cobalamine Cobalamine, B12 Izomeration, transfer of methyl group
8
Cofactor Group transferred
Adenosine triphosphate (ATP) Phosphate group
S-adenosylmethionin (SAM) Methyl group
Coenzyme Q Electrons
Cytidine triphosphate (CTP) Diacylglycerols and lipid head groups
Glutathione Electrons
Heme Electrons
Tetrahydrobiopterin Oxygen atom and electrons
9
Classification of cofactors by reactions that
help catalyze

• Cofactors of oxidoreductases
• NAD(P),
• FAD,
• cytochromes (containing heme),
• Fe-S complexes
• Cofactors that transport one carbon residues
• tetrahydrofolate,
• vitamin B12,
• S-adenosylmethionin,
• biotin (cofactor of carboxylases)
• Cofactors that transport acyl group
• lipoic acid (prosthetic group of PDH, a-KGDH),
• HSCoA,
• pyridoxal phosphate (transaminases)

10
Cofactors for oxidoreductases

• Nikotinamide adenine dinukleotide (NAD)
• Nikotinamide adenine dinukleotide phosphate
  (NADP)
• Precursor is niacin (vitamin B3)
• Flavin adenine dinucleotide (FAD)
• Flavin mononucleotide (FMN)
• Precursor is riboflavin (vitamin B2)
• Coenzyme Q
• Cytochroms Fe3 e- ? Fe2 (cyt a1, cyt a3, cyt
  b, cyt c)
• Cu2 e- ? Cu (cyt a)

11
NAD (NADP)
12
The redox reaction of NAD

• In metabolic oxidation of nutrients ? NAD
  accepts or donates electrons in redox reactions.
• RH2 NAD ? NADH H R
• Reactions involve the removal of two hydrogen
  atoms from the reactant (R), in the form of a
  hydride ion (H-), and a proton (H).
• The proton is released into solution, the
  reductant RH2 is oxidized and NAD reduced to
  NADH by transfer of the hydride to the
  nicotinamide ring.

13
FAD, FMN
14
Vitamin B2
FMN ? ATP-dependent phosphorylation of
riboflavin FAD ? further reaction with ATP in
which its AMP moiety is transferred to FMN.
15
Flavin Mechanisms

• Flavins are one- or two-electron transfer agents
• Name "flavin" comes from Latin flavius for
  "yellow
• The oxidized form is yellow, semiquinones are
  blue or red and the reduced form is colorless

16
Reduction and reoxidation of FMN and FAD
17
Some enzymes containing FAD, FMN

• acyl-CoA dehydrogenase FAD
• succinate dehydrogenase FAD
• glycerol-3-phosphate dehydrogenase FAD
• NADH reductase (complex I) FMN

18
Koenzym Q

• Component of the electron transport chain
• Participates in aerobic cellular respiration,
  generating energy in the form of ATP
• Coenzyme Q in human mitochondria is CoQ10 (Q
  the quinon head , 10 the number of isoprene
  repeats in the tail
• Hydrofilic head is movable, it transferres
  electrons, polyisoprene tail is anchored in the
  membrane..

Reduced form
Oxidized form
19
Cofactors of transferases

• Adenosine triphosphate (ATP) ? phosphate
• Guanosit triphosphate (GTP) ? phosphate
• Pyridoxal phosphate (PLP) ? -NH3
• (pyridoxine, vitamin B6)
• Tetrahydrofolate (THF) ? C1-fragment
• (folic acid, vitamin B6)
• Coenzyme A (CoA, HS-Co-A) ? acyl
• S-adenosylmethionin (SAM) ? active methyl

20
ATP
P - P - adenosin (ADP)
inorganic phosphate (Pi)
P - adenosin (AMP)
inorganic pyrophosphate (PPi)

• ATP transports chemical energy within cells for
  metabolism
• Substrate in signal tranduction pathways by
  kinases (transferases) that phosphorylate
  proteins and lipids
• Energy stored in ATP may be released upon
  hydrolysis of the phosphoanhydride bonds
• Gama phosphate group has a higher energy of
  hydrolysis than either the a- or ß-phosphate

21
Pyridoxalfosfát (PLP)

• Prosthetic group of aminotransferases
• Coenzyme in all transamination reactions, and in
  some decarboxylation and deamination reactions of
  amino acids.

Pyridoxine (vit. B6)
Pyridoxal-5-phosphate (PLP)
Pyridoxamine-5-phosphate
22
Thiamine pyrophosphate (diphosphate), TPP

• TPP is an active form of vitamin B1 (first
  discovered vitamin).
• TPP is consist of substituted rings (pyrimidine,
  thiazole). Highly hydrophilic substance.
• TPP catalyses the reversible cleavage of a
  substrate compound at a C - C bond connecting a C
  O group to an adjacent reactive group (usually
  a carboxylic acid or an alcohol)
• TPP transfers two-carbon residues in the
  cytoplasmic transketolase reaction in pentose
  phosphate pathway
• Oxidative decarboxylation reaction of a-ketoacids
  and formation of aldehydes (pyruvate ?
  acetaldehyde), cofactor of multienzyme complexes
  (PDH, a-KGDH)

23
Tetrahydrofolate (THF)

• Active form is tetrahydrofolate
• Folates are donors of 1-C units for all oxidation
  levels of carbon except that of CO2
• THF is coenzyme of transferases. N5,N10-THF
  transfers one carbon units, methylene or
  methenyl
• This reaction is part of the synthesis of
  nucleotides and nucleic acids.

24
Tetrahydrofolate (THF)
25
Coenzyme A

• Coenzyme A functions as an acyl group carrier
• A thiol can react with carboxylic acids to form
  thioesters.
• It assists in transferring fatty acids from the
  cytoplasm to mitochondria
• Acetyl coenzyme A (acetyl-CoA) is a macroergic
  compound effective in a variety of metabolic
  reactions (b-oxidation of fatty acids,
  biosynthesis of lipids)

26
S-adenosylmethionin
27
Biotin

• Condensate of urea and thiofene the rest of
  valeric acid
• Coenzyme in carboxylation reaction
• Prosthetic group of acetyl-CoA carboxylase and
  other ATP-dependent carboxylases
• Covalently attached to apoenzyme bound to e-amino
  group of lysine.

28
Lipoic acid

• Saturated fatty acid 8 carbons)
• Carbons 6, 7 a 8 constitute heterocycle with two
  atoms of sulfur.
• Is prosthetic group of enzymes, transmits
  hydrogen and and acyl group .
• Covalently attached to apoenzyme - binds an amide
  bond to e-amino group of lysine, why it is also
  called lipoamid.
• Oxidation and decarboxylatio of a-ketoacids
  (pyruvate dehydrogenase, a-ketoglutarate
  dehydrogenase)
• Antioxidant (reduced form is regenerated vit. C
  and vit. E).

29
Vitamin A and vision

• Vit. A is necessary to form rhodopsin (in rodes)
  and iodopsin (in cones) - visual pigment.
• Retinaldehyd is a prosthetic group of
  light-sensitive opsin protein.
• In the retina, all-trans-retinol is isomerized to
  11-cis-retinol ? oxidized to 11-cis-retinaldehyd,
  ? reaction with opsin (Lys) ? to form the
  holoprotein rhodopsin.
• Absorption of light ? conformation changes of
  opsin ? photorhodopsin.

30
Vitamin K
Vitamin K1
Fyloquinone

• Vitamin K1 (fyloquinone) plant origin
• Vitamin K2 (menaquinone) normally produced by
  bacteria in the large intestine
• K1 a K2 are used differently in the body
• K1 used mainly for blood clothing
• K2 important in non-coagulation actions - as
  in metabolism and bone mineralization, in cell
  growth, metabolism of blood vessel walls cells.

Vitamin K2
Menaquinone
Synthetic derivatives of Vit.K
31
Vitamin K - function

• Cofactor of liver microsomal carboxylase which
  carboxylates glutamate residues to
  g-carboxyglutamate during synthesis of
  prothrombin and coagulation factors VII, IX a X
  (posttranslation reaction).
• Carboxylated glutamate chelates Ca2 ions,
  permitting the binding of blood clotting proteins
  to membranes.
• Forms the binding site for Ca2 also in other
  proteins osteocalcin.

32
(No Transcript)
33
Tocoferol, vitamin E

• alpha-tocopherolchinon formed by oxidation from
  a-tocopherol is a cofactor in the synthesis of
  mitochondrial unsaturated fatty acids.

Chromanol ring hydrophobic aliphatic side
chain
34
Vitamin E as antioxidant

• Stops free radical reactions (peroxyl radicals
  ROO? , oxygen radicals HO?, lipoperoxid radicals
  LOO?). Chromanol ring with OH group ? uptake
  radicals
• ?-TOH ROO? ?-Toc? ROOH
• ROO? ?-Toc? ?TocOOR

35
Vitamin C

• Hydroxylation of proline and lysine (collagen)
• Synthesis of collagen prolylhydroxylase,
  lysylhydroxylase a lysyloxidase containe Fe2 and
  ascorbate as cofactors.
• Proline (lysine) a-ketoglutarate O2 ?
  4-hydroxyproline (hydroxylysine) CO2
  succinate
• Metabolism of Tyr in brain
• Fe mobilization from spleen
• May prevent the toxic effects of some metals
• Ameliorates allergic responses
• Can stimulate the immune system
• Vitamin C is a reasonably strong reducing agent
• It functions as an electron carrier

36
Vitamin C as antioxidant
37
Ions as a cofactors
Ion Examples of enzymes containing this ion
Cu2 Cytochromoxidase, catalase
Fe2 a Fe3 Cytochroms, hydroxylases
Mg2 Glucose-6-phosphatase, hexokinase, DNA-polymerase,
Mn2 Arginase
Zn2 Alcohol dehydrogenase, DNA polymerase, carboanhydrase
Se Glutathionperoxidase
FeS proteins (Fe2S2) NADH dehydrogenase, succinate dehydrogenase
38
Ferredoxins

• FeS proteins ferredoxins - Fe2S2, Fe4S4
  electron transfer
• redox reaction in mitochondrial respiratory
  chain (complex I and complex II)
• NADH dehydrodenase, koenzym Q - cytochrom c
  reductase, succinate koenzym Q reductase

39
Metalloporphyrins - heme

• Organic, cyclic compounds derived from the
  tetrapyrrole - porphyrin - four pyrrol rings are
  linked with methylene bridges.
• Complexes with metal ions
• heme Fe, oxygene transport
• chlorophyl Mg, photosynthesis

40
Metalloporphyrins - cobalamin (vitamin B12)

• Chemically most complex vitamin
• Structurally similar to heme - complex of organic
  compounds, atom within the molecule is Co,
• In man there are two metabolically active forms
  methylkobalamin and adenosylkobalamin.
• Cobalamin catalyses two reactions
• cytoplasmic methylation of homocystein to
  methionin.
• mitochondrial methylmalonyl-CoA mutase
  (methylmalonyl-CoA ? sukcynyl-CoA) needs deoxy
  adenosylkobalamin.