Cofactors and coenzymes · 2019-03-21 · Cofactors and coenzymes. 1 Many enzymes require the...
Transcript of Cofactors and coenzymes · 2019-03-21 · Cofactors and coenzymes. 1 Many enzymes require the...
Cofactors and coenzymes
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Many enzymes require the presence of small, non-proteinaceous molecules (or ions) termed cofactors for function
Cofactors can be divided into three different groups: Coenzymes (sometimes called cosubstrates) Prosthetic groups Metal ions
Definition of a coenzyme (IUPAC): a low-molecular-weight, non-protein organic compound that is loosely attached, participating in enzymatic reactions as a dissociable acceptor/donor of chemical groups or electrons
In contrast: a prosthetic group is defined as a tightly bound, nonpolypeptide unit in a protein (IUPAC) – e.g., [Fe4S4] clusters, heme groups
Coenzymes are continuously recycled as part of an organism’s metabolism
Based on the precursor molecule, coenzymes are classified into two types Vitamin-based coenzymes (e.g., NADH, FADH2, CoA, TPP) Non-vitamin-based coenzymes (e.g., ATP, CoQ)
Vitamin-derived coenzymes
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These coenzymes are derivatives of vitamins (most cases) or the vitamin itself (vitamin C and vitamin K)
Coenzyme Precursor Function
NAD(P)+/NAD(P)H Vit B3: Niacin (nicotinic acid)
Primarily redox reactions; hydride transfer
Thiamine pyrophosphate (TPP)
Vit B1: Thiamine Carbohydrate metabolism
Flavin coenzymes Vit B2: Riboflavin Electron carrier in redox reactions
Coenzyme A (CoA) Vit B5: Pantothenicacid
Acyl group carrier
Pyridoxal phosphate (PLP)
Vit B6: pyridoxine Amino acid metabolism
Tetrahydrofolate Vit B9: folate One-carbon unit carrier
Vitamin-derived coenzymes
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Coenzyme Precursor Function
Cobamide Vit B12: Cyanocobalamin
Intramolecular arrangements
Biotin Vit H: Biotin CO2 fixation reactions
L-Ascorbate Vit C: Ascorbic acid Hydroxylations,decarboxylations, amidations
Phylloquinone / menaquinone
Vit K: Pantothenicacid
Carboxylation of Glu residues
Lipoamide Lipoic acid Transfer of electrons and acyl groups
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Nicotinamide coenzyme
First coenzyme to be discovered (by Harden & Young in 1906)
The coenzyme is derived from nicotinamide, which itself is derived from nicotinic acid (also known as niacin; Vit B3)
Nicotinamide coenzymes can be unphosphorylated (NAD+) and phosphorylated (NADP+)
(nicotinamide adenine dinucleotide; NAD+) (NADP+)
niacin
nicotinamide
AMP moiety
ribose
Note that NAD(P)+ is actually an anion!
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Nicotinamide coenzyme
Can exist in two redox states: Oxidized: NAD(P)+
Reduced: NAD(P)H
NAD+ facilitates oxidative reactions (catabolic)
NADPH facilitates reductive reactions (anabolic; i.e., biosynthetic)
Free cytoplasmic NAD+/NADH ratio is ca. 700 Therefore: The NAD+/NADH system participates in oxidative reactions
The NADP+/NADPH ratio is ca. 0.005 Therefore: The NADP+/NADPH system participates in reductive reactions
hydride-carrying C4
midpoint potential: -320 mV
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Functions of NAD+/NADH
Coenzyme in (oxidative) redox reactions: Isocitrate dehydrogenase (isocitrate ↔ α-ketoglutarate) Glutamate dehydrogenase (Glu ↔ α-ketoglutarate) Alcohol dehydrogenase (ethanol ↔ acetaldehyde) Glyceraldehyde-3-P dehydrogenase (glyceraldehyde-3-P ↔ 1,3-bisphospho-
glycerate) Lactate dehydrogenase (lactate ↔ pyruvate)
Monomer of homotetrameric human muscle lactate dehydrogenase (pdb: 1I10)
Rossmann fold (many NAD+ requiring enzymes have this type of fold)
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Structure of horse liver alcohol dehydrogenase (mutant with NAD+ and trifluoroethanol) Shows the proximity of the nicotinamide moiety and the substrate analog
Pdb: 1AXG Zn2+: magenta spheres (catalytic and structural zinc sites)
structural zinc
Mechanism: Theorell-Chance (1. + NAD+; 2. + EtOH; 3. – CH3CHO; 4. – NADH)
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NAD+ also plays a role in non-redox reactions:
Post-translational modification (e.g., in ADP-ribosyltransferase, which transfers the ADP-ribose moiety to proteins)
Cell signaling: Synthesis of cyclic ADP-ribose (involved in Ca2+ signalling) by ADP-ribosyl cyclases
Regulation of transcription (NAD+ accepts an acetyl group from histones → mediated by NAD+-dependent deacetylases, called sirtuins)
Cyclic ADP ribose
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Functions of NADP+/NADPH
NADPH is a coenzyme in (reductive) redox reactions
Used in anabolic reactions (Calvin cycle, lipid synthesis, nucleic acid synthesis)
Example Photosynthesis: NADPH is produced from NADP+ by ferredoxin:NADP+ reductase (final step
of the light reactions) Calvin cycle (dark reactions): NADPH is required to provide reducing
equivalents, essentially converting CO2 to glucose In the Calvin cycle, NADPH is a coenzyme for glyceraldehyde-3-P
dehydrogenase (1,3-bisphosphoglycerate → glyceraldehyde-3-P)
NADPH is also used to: reduce nitrate to ammonia (plants) eliminate ROS (by reduction of oxidized glutathione (GSSG → GSH)
mediated by glutathione reductase) elongate fatty acids (coenzyme in fatty acid synthase) Drive the rate-limiting step in cholesterol biosynthesis (coenzyme in HMG-
CoA reductase [HMG = 3-hydroxyl-3-methyl-glutaryl] → target for statins, drugs that lower cholesterol)
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Enzyme assays
259 nm
339 nm
Spectrophotometry: generation/consumption of NAD(P)H can be measured at 339 nmε259 = 16,900 M-1 cm-1
ε339 = 6,620 M-1 cm-1
Fluorescence spectroscopy: in aqueous solution, NADH fluoresces (emission @ 460 nm), whereas NAD+ does not. The fluorescence signal changes when the coenzyme is bound to a protein (measurement of Kd, enzyme kinetics, redox state of living cells).
A: NADPHB: BLCC: NADPH +
BLC
BLC = bovine liver catalase (BLC)
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Flavin coenzymes
Derived from riboflavin (Vit B2) Flavoenzymes are electron carriers and contain either:
FMN (Flavin mononucleotide) or FAD (Flavin adenine dinucleotide)
isoalloxazine ring
ribitol
Riboflavin = FMN without the phosphate group
FMN is the blue part of the structure
FAD is an electron acceptor in reaction of the type
Human genome:90 flavoproteinsca. 85% FAD-dep.ca. 15% FMN-dep.
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FAD (and also FMN) can exist in multiple redox states, but the following two are the most common:
coloured colourless
Examples of flavoenzymes: Succinate dehydrogenase (succinate → fumarate) FAD Glutathione reductase (GSSG → GSH) FAD Glucose oxidase (glucose → glucono-lactone) FAD Amino acid oxidase (amino acid → ketoacid + NH3) FAD Aldehyde oxidase (aldehyde → carboxylic acid) FAD p-hydroxybenzoate hydroxylase (p-OHB → 3,4-diOHB) FAD 2-nitropropane dioxygenase (2-nitropropane + O2 → acetone + nitrate) FMN
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A glutathione reductase with NADPH and FAD bound (pdb: 2X99)
Side-view
Mechanism of electron transfer in glutathione reductase
Perfect alignment of the nicotinamide ring with the central ring of the isoalloxazine moiety
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Mechanism of p-hydroxybenzoate hydroxylase (a FAD-dependent monooxidase)
FAD-OOH FAD-OH
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Coenzyme A
Derived from pantothenic acid (Vit B5)
Structure of CoA:pantothenic acid
CoA functions as an acyl carrier (A = acyl), with the reactive part being the thiol moiety
General mechanism:1. CoA reacts with a carboxylic acid to form a thioester2. A nucleophile attacks the carbonyl carbon atom, and becomes acylated
pantothenic acid β-mercapto-ethanolamine
3’-phosphoadenosine
diphosphate (anhydride)
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CoA-activated acyl groups: Acetyl-CoA Propionyl-CoA Acetoacetyl-CoA Coumaroyl-CoA Malonyl-CoA Succinyl-CoA Butyryl-CoA
Reactions catalyzed by CoA: In citrate synthase:
p-coumaric acid
Malonyl-CoA
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Mechanism of citrate synthase
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Acetyl-CoA carboxylase (requires biotin and ATP) is the starting point for fatty acid synthesis
CoA in fatty acid synthases (cytosolic):
Fatty acid chain elongation
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Relationship between acyl carrier protein (ACP) and coenzyme A
ACP is a 77-residue protein, and can be regarded as a large prosthetic group (macro-CoA)
In the initiation of elongation, acetyl-CoA and malonyl-CoA react with ACP to form acetyl-ACP and malonyl-ACP and CoA (catalyzed by transacylases)
serine
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Tetrahydrofolate
Folic acid/folate (from Latin: folium – leaf) is also known as vitamin B9 and participates in biochemical reactions as tetrahydrofolate (an essential one-carbon carrier)
pterin
methylene bridge
PABA L-Glu
Structure of THFPurple arrows indicate the positions of the 4 extra hydrogen atoms
Note: folic acid can have a chain of one or more L-Glu residues
PABA = p-aminobenzoic acid
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Tetrahydrofolate
THF functions as a 1-carbon carrier for all oxidation levels of carbon except CO2
A variety of carbon-containing groups can be attached to N5 nitrogen atom of the pterin ring and on the N10 nitrogen atom of the PABA moiety
Overall, there are six forms of THF, existing in different oxidation states (+2, 0, -2), which can carry five different one-carbon units (see below)
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Structures of the one-carbon units attached to THF
THF and its derivatives participate in a variety of synthetic reactions:
Synthesis of methionine
Synthesis of inosine monophosphate (IMP) → synthesis of purine nucleotides
Thymidine phosphate synthesis
THF is important in: DNA synthesis and cell division Prevention of anemia Reducing the risk of stroke Prevention of congenital malformations in the fetus Improving fertility in women (oocyte maturation,
implantation, placentation) and men (spermatogenesis)
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Biotin
Biotin (Vitamin B7 or H) is required for the formation of fatty acids and glucose, and assists in the metabolism of carbohydrates, fats and proteins
Biotin is used for carboxylase-catalyzed CO2 transfer reactions, where the CO2group is usually donated by HCO3
-
Hence, biotin is a carboxyl carrier.
The structure consists of a bicyclic ring (tetrahydrothiophene attached to urea) with a valeric acid side chain(the N-containing ring is a ureido ring)
CO2 binding site
Binds to Lys residue of apocarboxylases (via amide bond)
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Examples of reactions catalyzed by enzymes requiring biotin:
Pyruvate carboxylase (HCO3- + pyruvate + ATP → oxaloacetate + ADP +Pi)
Propionyl-CoA carboxylase (HCO3- + propionyl-CoA + ATP → (S)-methylmalonyl-
CoA + ADP +Pi)
Acetyl-CoA carboxylase (HCO3- + acetyl-CoA + ATP → malonyl-CoA + ADP +Pi)
Urea carboxylase (HCO3- + urea + ATP → N-carboxy urea + ADP +Pi)
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General mechanism:
Carboxylase reactions proceed in two steps:
1. Activation of bicarbonate by ATP and transfer to biotin
2. Transfer of carboxyl group to substrate
Example mechanism:Propionyl-CoA carboxylase
Carbonic phosphoric anhydride
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Carboxylated biotin
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Cobamide coenzymes
Cobamide coenzymes are derived from cyanocobalamin (Vitamin B12)
The cobalt ion is bound to the N atoms of a corrin ring and dimethylbenzimidazole
Physiologically relevant forms of cobalamin
Coenzyme B12
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Cobalamine-dependent enzymes catalyze a variety reactions involving activated alkyl groups. It is found in: Isomerases (mutases) → especially 1,2-shifts at saturated carbon centres Dehydratases Lyases (C-C, C-O, C-N bond cleavage) Ribonucleotide reductase (bacterial) Methyltransferases
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Mammals possess only two cobalamin-dependent enzymes: Methylmalonyl-CoA mutase (Coenzyme B12-dependent) Methionine synthase
Coenzyme B12-dependent processes use an adenosyl radical !
Adenosyl radical
General mechanism:
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Methylmalonyl-CoA mutase
Mechanism:
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Methionine synthase (MS)
MS catalyzes the methylation of homocysteine to methionine
MS is a methyltransferase requiring methylcobalamin and N5-methyl THF as coenzymes
Besides cobalamine, SAM is a methyl donor in biosynthetic reactions!
SAM is a non-vitamin derived coenzyme
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Mechanism of methionine synthase
Methylcobalamin (Co3+) methylates homocysteine resulting in a Co+ supernucleophile
The supernucleophile abstracts the methyl group from N5-CH3-THF, thereby regenerating methylcobalamin
in MS, the benzimidazole attached to the corrin ring is displaced by an enzyme His residue
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Coenzyme Q10
Coenzyme Q10 (CoQ10) or ubiquinone is a non-vitamin-derived coenzyme
It is found in membranes of the ER, peroxisomes, lysosomes, and in the inner mitochondrial membrane (part of the electron transport chain)
isoprenyl units
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Electron transport chain
Organs (liver, heart) with the highest energy requirements have the highest CoQ10 concentrations
Applications of CoQ10: Migraine headaches Cancer treatment Neurodegenerative diseases Blood pressure regulation Lifespan extension (in rats)