Online Test 2 Flashcards
Number of stages of metabolism cycle
Stage 1, 2, 3
Stage 1
Breakdown macromolecules into building blocks
Stage 2
Oxidation of stage 1 products to Acetyl CoA, limited energy production
Stage 3
Oxidation of Acetyl CoA to CO2, H2O and energy
Catabolism
- Breakdown of complex organic molecules to simpler species to produce ATP
- converging pathway
- Chemical oxidation & reduced cofactors NADH, NADPH, FADH
Anabolism
- Biosynthesis of complex organic molecules using energy from ATP, NADH etc
- diverging pathway
- Chemical reduction & oxidized cofactors NAD+, NADP+
Monosaccharides
Glucose
Fructose
Disaccharides
Maltose (glucose + glucose)
Sucrose (glucose + fructose)
Lactose (glucose + galactose)
Polysaccharides
Fibre
Starch
Glycogen
breaking down of carbohydrates (enzyme)
Starch is digested by amylase
Amylase is found in the mouth, pancreas
Small intestines have maltase, lactase, sucrase (requires H2O to hydrolise and break down)
Hydrolysis of di/polysaccharides
Glucose is absorbed and transported in the intestinal walls to peripheral tissues.
1/3 to skeletal and heart muscles for energy production & storage
1/3 to brain for aerobic energy production
1/3 to liver for glycogen storage
Dietary glucose can be used for synthesis of other carbs
Features of Glycolysis
- First step for glucose oxidation for further energy production in citric acid cycle and oxidative phosphorylation
- Universal pathway in flora and fauna
- Anaerobic
- Turns to pyruvate anaerobically with small amounts of energy produced (ATP, NADH)
- Takes place in cytoplasm due to enzyme location
- Net ATP: 2 (consumes 2 ATP, produces 2 NADH, 4 ATP
Pathways of Pyruvate
metabolic options:
lactate fermentation: anaerobic
ethanol fermentation: anaerobic
Acetyl CoA: aerobic
-oxygen availability determines outcome of pyruvate
Pyruvate to Acetyl CoA (3 chemical reactions)
Occurs at pyruvate dehydrogenase complex
- Decarboxylation (loss of CO2)
- Oxidation of C2 keto group to a carboxyl group
- Activation by linkage to Acetyl CoA through a thioester bond
Pyruvate hydrogenase complex formula
Pyruvate + CoASH + NAD = Acetyl CoA + NADH + H +CO2
Fermentation of pyruvate
- Anaerobic
- Used to make NAD when there is insufficient O2
- NAD must be regenerated from NADH or glyclosis stops
Lactate fermentation
pyruvate + NADH + H = Lactate + NAD
anaerobic of pyruvate regenerated NAD
oxygen debt collects to oxidize NAD
Ethanol fermentation
Occurs in yeast in bacteria, used to produce NAD and reduce pyruvate in the cytoplasm
Overall glycolysis
Glucose + 2 ATP + 2 phosphate + 2 NAD = 2 Pyruvate + 2 NADH + 2 H2O + 2 ATP
Features of Citric Acid Cycle
- Aerobic
- Acetate combines with oxaloacetate to form citrate when Acetyl CoA enters cycle
- acetate is oxidised to 2 CO2 (during citrate to oxaloacetate conv)
- at the end of cycle, we have oxaloacetate again, and 1GTP/ATP, 3 NADH and 1 FADH2
- oxaloacetate is reused
Citric Acid Cycle Steps
- Acetyl CoA (2C) enters the citric acid cycle, joins oxaloacetate (4C)
- Acetyl CoA + oxaloacetate = citrate (6C)
- Citric acid is broken down, modified in a stepwise direction. H ions and CO2 is produced (5C)
- H ions are picked up by NAD and FAD
- The process forms oxaloacetate (4C) again
Summary:
Citric acid cycle can produce many ATP molecule from one Glucose molecule
Citric Acid Overall formula
Acetyl CoA + 3NAD + ADP
3NADH + 3H + FADH + ATP/GDP + CoA + 2CO2
NADH + FADH leftover
If O2 is present, the citric acid cycle continues, and the electrons of NADH & FADH are delivered via electron transport system for oxidative phosphorylation (to produce ATP)
Amount of energy at different stages
Glycolysis: -2ATP, +2NADH, +4ATP
Pyruvate to Acetyl CoA: +2NADH (since 1 glucose = 2 pyruvate)
Acetyl CoA to citric acid cycle: +6NADH, +2FADH, +2ATP
Main source of fatty Acid
- Dietary triacylglycerol
- Triacylglycerol synthesised in the livers
- Triacylglycerol stored in fat cells
Digestion of lipids
- hydrophobic
- Lipase in duodenum and saliva
- Bile to further emulsify the small globules of fat in the duodenum (increase surface area to volume ratio)
- Bile is produced in the liver and stored in the gall bladder, and enters GI tract at the start of small intestines
- Pancreatic lipase is used to hydrolyse fats, but needs assistance of colipase protein that binds to surface of lipid droplet
Fats + H2O +lipase = Fatty acids + monoacylglyceride
absorption of lipids
- fatty acids and monoacylglycerol pass through small intestine wall
- reassembled into triacylglycerols
- lipoprotein is added and chylomicrons are produced, which are passed through the lymph node system
Fate of fatty acids
- stored as triacylglycerides in adipose tissue
- b-oxidized for ATP (yields acetyl CoA)
Fatty acid entry
- immediately activated by linking them by thioesters to CoASH
- labels them for energy degradation since activation is coupled with their transport through the mitochondrial membrane
b-oxidation of fatty acids
- fatty acids highly reduced, oxidized by NAD & FAD
- fatty acids metabolised to acetyl CoA-oxidized by NAD & FAD in citric cycle
- NADH & FADH carry electrons to electron transport chain (ETC) to power ATP production
Lipid catabolism steps
- activation step
2. b-oxidation
Activation step (Lipids)
- Convert fatty acid into fatty acyl CoA, requires energy (2 ATP)
- Fatty acyl enters mitochondria for further degradation
b-oxidation step (Lipids)
not in lec video
- Fatty acids are recycled through the same 5 step process:
oxidation of C-C, addition of H2O, oxidation of -OH group, cleavage of C-C (double-check, only states 4 on slides)
-Spiral pathway, each has 4 enzyme-catalysed steps resulting in 1 Acetyl CoA molecule, and fatty acid reduced by 2C each time
Amino acids consists of
amine and carboxylic acid group
Acid chain names
Amio acid chain: peptide
longer chain: polypeptide
Types of proteins
Simple vs Conjugated,
Globular vs Fibrous
Protein Digestion
Pepsin found in stomach, with acidic environment (HCL)
Pancreatic enzyme: trypsin, chymotrypsin in small intestines
enzyme dipeptidase in small intenstines
peptidase helps break down proteins
polypeptides > peptides > amino acid, absorbed through capillary blood and transported to liver
Protein Catabolism: Urea Cycle
- happens in the liver cytoplasm and mitochondrion
- involved enzymatic reaction
Semiconservative nature of DNA replication
- Base pairing double helical structure of DNA facilitates replication
- DNA is semi-conservative: each new DNA double helix contains one parent one daughter strand
- replication mode used by prokaryotes and eukaryotes
functions of the different classes of DNA polymerase
DNA Pol I: removes and replaces primper
DNA Pol II: DNA repair; restarts replication after damaged DNA halts synthesis
DNA Pol III: Elongates DNA
DNA Pol IV: DNA repair
DNA Pol V: DNA repair; translesion DNA synthesis
identify and describe the stages of bacterial DNA replication
Three major steps:
Initiation: σ subunit recognises the promoter and RNA pol binds to the DNA
Elongation: RNA pol advances in the 3′–5′ direction along the template strand, synthesising RNA in the 5′–3′ direction
Termination: RNA pol encounters a transcription terminator, releases the completed RNA, and dissociates from the DNA
contrast bacterial and eukaryotic DNA synthesis
Bacteria:
first amino acid is fMet
contains approx. 10,000 ribosomes
70s ribosome consists of 50S and 30S subunit
Eukaryotic:
first amino acid is Met
contains over 50,000, some amphibian eggs over a million ribosomes for high protein synthesis
80S ribosome consists of 60S and 40S subunit
how many bps must be replicated for human genome
3 x 10^9
Meselson-Stahl experiment
Medium: 15NH4Cl to 14NH4Cl
isotope: 15N
15N reaches equilibrium in density gradient closer to the bottom than the less dense 14N
Prokaryotic origins of replication
- E. coli: replication begins from fixed origin (Ori C), then proceeds bidirectionally
- Ori C 245 base pair long
- moving fork occurs at both ends of replication piece
- End of replication point called terminus
DNA polymerases
Enzyme catalyzing chain elongation (polymerization) reactions using dNTPs as substrate molecules
- Identified & purified by Kornberg in 1950
- DNA polymerase links innermost 5’ phosphate of incoming dNTP to 3’ hydroxyl at end of the growing chain, forming phosphodiester bond
- energy for linking process comes from hydrolysis of phosphate bonds in dNTPs
E.coli purified enzyme
DNA Pol I, II, III, IV, V
DNA Pol I activities
- 5’ - 3’ polymerase activity: catalyzes chain elongation
- 3’ - 5’ exonuclease activity: removes mismatched base
- 5’ - 3’ exonuclease activity: degrades dsDNA
DNA Pol III
responsible for polymerization activity
Active form: holoenzyme, contains 10 different polypeptide sub units, molecular weight 900 kDa
Subunits of DNA Pol III (not tested)
α (alpha) 5´ – 3´ ε (epsilon) 3´ – 5´ θ (thêta) γ (gamma) δ (delta) δ´ χ (chi) ψ (psi) β (beta) τ (tau)
Mechanistic issues of replication
- The helix must be stably unwound to expose the template strands
for replication - The tension created by unwinding must be minimised
- Primers must be synthesised for DNA pol III to extend
- DNA pol III needs to synthesise DNA both continuously and
discontinuously on opposite strands - Primers must be removed and replaced, and gaps sealed, before
replication is complete - Any errors introduced during replication must be corrected
Summary of DNA replication
- replicated from replication fork that proceeds from a single origin in prokaryotes
- replicated by polymerase that catalyzes template-directed chain elongation from DNA or RNA primer
(Possess 5 DNA polymerase, DNA Pol III being major synthetic enzyme)
Double Helix unwinding (pt. 1)
- initiated when subunits of the initiator protein (DnaA) bind to 9-mer sequences in oriC
- facilitates subsequent binding of helicase (DnaB & DnaC) proteins that further open and destabilise the helix
- Helicases use energy from ATP to break hydrogen bonds and denature the double helix
- The open confirmation is stabilised by single-strand-binding proteins (SSBPs)
Double Helix unwinding (pt. 2)
As unwinding proceeds, a coiling tension is created ahead of the replication fork, often producing supercoiling
- This supercoiling is relaxed by DNA gyrase, a type of DNA topoisomerase
- DNA gyrase makes single- or double-stranded cuts and catalyses localised movements that undo the twists and knots created during supercoiling
- Cut strands are then resealed
Priming DNA synthesis
- Once a small portion of the helix is unwound, the initiation of synthesis may occur
- DNA pol III requires a primer with a free 3´ end in order to elongate a polynucleotide chain
- DNA synthesis is initiated when DNA pol III adds deoxyribonucleotides to the 3´ end of the RNA primer
- A short 5- to 15-mer of RNA is synthesised on the DNA template by a form of RNA polymerase called primase
- Primase is able to catalyse de novo RNA synthesis
Discontinuous synthesis
- The two strands of the double helix are antiparallel: one runs in a 5´–3´ direction (lagging strand), while the other runs in a 3´–5´ direction (leading strand)
- DNA pol III synthesises DNA only in the 5´–3´ direction
- Synthesis along an advancing replication fork occurs in one direction on one strand and in the opposite direction on the other
Leading vs Lagging strand
Leading Strand: only one strand can serve as a template for continuous DNA synthesis
Lagging Strand: The other strand serves as a template for discontinuous DNA synthesis
Okazaki fragments
- Evidence supporting discontinuous synthesis was first provided by Reiji and Tuneko Okazaki in 1968
- The Okazakis showed that when bacteriophage DNA is replicated in E. coli, some of the newly formed DNA is present as small fragments containing 1000–2000 nucleotides, each containing an RNA primer as part of the fragment
- Okazaki fragments are joined into contiguous strands as synthesis proceeds
- joined by DNA ligase, which catalyses the ATP-dependent formation of phosphodiester bonds and thus seals the nicks between the fragments
Discontinuous synthesis (DNA Pol I)
- responsible for removing the RNA primers and replacing the ribonucleotides with deoxyribonucleotides (DNA)
Discontinued Synthesis (Okazaki fragments)
joined with DNA ligase which catalyzes ATP-dependent formation between phosphodiester bonds, sealing the nicks between the fragments
Concurrent Synthesis
- DNA pol III dimer is able to process DNA templates on both strands in the same direction
- The lagging strand is looped so as to bring its orientation into line with the leading strand
- The β subunit of DNA pol III, or sliding clamp, prevents the core enzyme (α, ε and θ subunits) from falling off the DNA template as synthesis proceeds
Proofreading
- mismatched nucleotide is inserted every ~100,000 nucleotides
- allows DNA pol III to detect and excise mismatched nucleotides in the 3´–5´ direction, after which synthesis proceeds in the 5´–3´ direction
Replication components
Initiator protein: binds to OriC and separates strands of DNA to initiate replication
DNA helicase: unwinds DNA at replication fork
single-strand-binding-proteins (SSBPs): attach to single-strand DNA to prevent unwinding
DNA gyrase: moves ahead of the replication fork, making and resealing breaks in double strand DNA to release torque that accumulates as a result of unwinding at the replication fork
DNA ligase: joins Okazaki fragments by sealing nicks in the sugar-phosphate backbone of the synthesized DNA
DNA primase: synthesis of primers to provide 3’ -OH group for attachment of deoxyribonucleotides
DNA polymerase III: elongates new nucleotide strand using 3’ -OH provided by primase
DNA polymerase I: removes primers and replaces with DNA
Acetyl CoA
- from intermediary metabolism of carb, A.A, FA
- main use to convey C atoms to citric acid cycle to be oxidized for energy production
- chemically, is thioester between coenzyme A and acetic acid
- used in FA metabolism to provide energy
reduced coenzyme
- high energy to donate to redox reactions
- NADH, FADH, NADPH gained from oxidation reactions
Pathways for carbohydrates, proteins and fats
Carbohydrate (glucose/ glycogen): glycolysis
Protein (amino acid): urea cycle
Fats (fatty acid): b-oxidation
Aerobic or Anaerobic process
Glycolysis: Anaerobic Pyruvate to Acetyl CoA: Aerobic Citric Acid Cycle: Aerobic Electron transport: Aerobic Oxidative phosphorylation:
Location in cell
Glycolysis: Cytoplasm
Pyruvate to Acetyl CoA: Cytoplasm to mitochondria
Citric Acid Cycle: Mitochondrial Matrix
Purpose of Oxidative Phosphorylation
To convert oxidized coenzyme, i.e NADH, NADPH, FAPH2 to ATP
How many energy is produced at the end of the citric acid cycle from lipids (b-oxidation)
31 NADH and 15 FADH (129 ATP)
Ori C Sequence
GATCTNTTNTTTT
