Exam 3 Flashcards
What form is G6P stored as? Why?
Glycogen
Why:
- Because it is a form of glucose that will not cause cell damage/disrupt the osmotic balance because it’s non-osmotic
- It is easily mobilized to be converted back into glucose when energy is needed
Why can’t glucose be stored in high concentrations in the body?
Having a high [glucose] disrupts the osmotic balance of the cell –> causes cell damage and death
Glycogen is not as energy rich as fatty acids because it’s not as reduced. Why then, is fatty acid not used as energy storage instead of glycogen?
Because glycogen allows for controlled release of glucose –> allows blood-glucose levels to be maintained in between meals –> more constant energy levels
Define: CATABOLISM
Set of metabolic pathways that BREAK DOWN down molecules into smaller units to release energy.
Where is glycogen primarily stored in the body? Where else is it stored?
Primarily stored in…
1. Liver 2. Skeletal Muscle
Also stored in…
-Cytoplasm
What is the function of glycogen in the liver and in skeletal muscle?
Function: regulation of glycogen synthesis and degradation for…
Liver: Maintenance of blood-glucose levels
Skeletal Muscle: To fulfill its own energy needs
What are the steps of glycogen degradation?
- The release of glucose 1-phosphate (G1P) from glycogen
- Remodeling of glycogen substrate to allow further breakdown
- Conversion of G1P to G6P (glucose 6-phosphate) for further metabolism
What are the 3 possible outcomes that can result from glycogen degradation?
- Used as starting material (substrate) for glycolysis
- Conversion into free glucose for release into the bloodstream
- Processed by the pentose phosphate pathway to make NADPH and ribose derivatives
Where does the conversion of glycogen into free glucose mainly take place?
Liver
When does glycogen synthesis occur? What does it require?
- Occurs when [glucose] abundant
- Requires UDP-glucose (uridine disphosphate glucose)
What is UDP-glucose?
(uridine disphosphate glucose)
- Activated form of glucose
- Formed by RXN of UTP + G1P
What are the enzymes involved in glycogen degradation?
- Phosphorylase
- “debranching enzyme”
- Transferase
- 1,6-glucosidase
- Hexokinase
What is the function of PHOSPHORYLASE in glycogen degradation?
- Cleaves Glu residues from glycogen by adding orthophosphate (Pi) to make G1P
(i. e. G1P= a Glu residue (unit) that makes up glycogen molecule
What is the function of the “DEBRANCHING ENZYMES” in glycogen degradation?
To catalyze the remodeling of glycogen
Remodel needed: remove the 4 residues of an α-1,6 linkage/branch
Why: phosphorylase cannot work when these residues are present
What are the 2 “DEBRANCHING ENZYMES” used in glycogen degradation?
- Transferase
- 1,6-glucosidase
What is the function of “TRANSFERASE” in glycogen degradation?
Transfer a trisaccharide from the α-1,6 branch to the main chain
What is the function of “1,6-GLUCOSIDASE” in glycogen degradation?
Remove the last residue of the α-1,6 branch as glucose
What is the function of “HEXOKINASE” in glycogen degradation?
Catalyze the phosphorylation of Glu to give G1P.
What is PHOSPHORYLSIS?
Breaking a bond by adding orthophosphate (Pi)
From where on glycogen’s structure does phosphorylase remove the Glu residues from?
From the nonreducing ends of the glycogen molecule (i.e. THE ENDS WITH A FREE OH GROUP ON CARBON 4)
How is the activity of PHOSPHORYLASE regulated in glycogen degradation?
- Allosteric Effectors
- signal energy state of the cell
- Reversible Phosphorylation
- responds to hormones (ex. insulin, glucagon) - Dif. tissues have dif forms of phosphorylase
- respond dif to effects of 1 and 2
Describe the 2 conformations that phosphorylase switches between.
T Conformation (inactive state/phosphorylase b): residue loop is blocking active site (residue loops form the deep crevice where active site is located)
R Conformation (active state/phosphorylase a): active state is not blocked
- Uses phosphorylase kinase to switch conformations
- Active site=glycogen binding site
What are ISOZYMES?
Enzymes that differ in amino acid sequence but catalyze the same rxns
What controls the MUSCLE isozyme of phosphorylase?
-AMP and G6P allosterically control it
How:
-presence of high [ATP] and [G6P]–> T state (inactive)
-presence of high [AMP] –> R state (active)
Why: low [ATP]=need more energy=glucose mobilization
What is ALLOSTERIC CONTROL?
Inhibition/activation of an enzyme via binding of a regulator to a part of the enzyme that’s NOT the active site
How does exercise affect the conformation of the MUSCLE isozyme of phosphorylase?
Triggers hormone release –> triggers phosphorylation of T state –> now in active R state
What controls the LIVER isozyme of phosphorylase?
-allosterically controlled by [glucose]
How:
- high [glucose] –> T state (inactive)
- low [glucose] –> R state (active)
*not affected by [AMP] b/c [AMP] stays fairly constant in liver
What activates PHOSPHORYLASE KINASE?
- Phosphorylation
- Calcium ions
- Hormones
What is the function of PHOSPHORYLASE KINASE?
To switch phosphorylase between the T and R states.
When active: it activates phosphorylase (R state)
When inactive: phosphorylase=T state
What signals the need for glycogen degradation?
- Need for Glucose –> activates glucagon
2. Need for Energy –> activates epinephrine (adrenaline)
How does phosphorylase kinase get activated by PHOSPHORYLATION?
- Hormones signals trigger cAMP signal-transduction cascade
- cAMP phosphorylates inactive PKA
- PKA is now activated
- PKA phosphorylates β-subunit of phosphorylase kinase
- Phosphorylase kinase is now active
How does phosphorylase kinase get activated by CALCIUM IONS?
δ-subunit of phosphorylase kinase=Calmodulin
- Calmodulin=calcium sensor that stimulates many enzymes in eukaryotes
- Increase in [Ca2+] –> triggers calmodulin to activate phosphorylase kinase
In which subunit does the catalytic activity of phosphorylase kinase reside in?
γ-subunit
What is the function of PHOSPHOGLUCOMUTASE?
Converts G1P –> G6P
What is the enzyme that allows glucose to leave the liver?
Phosphoglucomutase
How:
1. phosphorylated serine residue located in G6P’s active site transfers a phosphoryl group to C-6 hydroxyl group of G1P to form G1,6P (glucose 1, 6-biphosphate)
- C-1 phosphoryl group of G1,6P is transferred to serine residue to form G6P and G1,6P
What are the chemical equations for glycogen degradation and synthesis?
Degradation:
(Glycogen,n+1) + Pi -> (Glycogen,n) +G1P
Synthesis: (Glycogen,n) + UDP-glucose -> (Glycogen,n+1) + UDP
What is UDP-GLUCOSE?
The activated form of glucose
What makes it “active”: hydroxyl group of glucosyl unit is esterfied to the diphosphate moiety of UDP
What enzyme is responsible for regulating GLYCOGEN SYNTHESIS?
Glycogen Synthetase
Function: Catalyzes the transfer of glucosyl residues from UDP-glucose to the nonreducing terminal residues of growing glycogen chain
*Glycogen synthase can only add residues onto chains that have at least 4 residues already on it
What enzyme is responsible for the BRANCHING that occurs in glycogen synthesis?
Transglycolase
-switches addition of glucosyl residues onto 1,4 carbon to addition onto 1,6 carbon
How: glucosyl residue is first transferred to enzyme and then to the 1,6-hydroxyl group of carbon
What does glycogen synthetase require for glycogen synthesis?
Glycogenin
-acts as the primer that glycogen synthetase
can add glucosyl units to
What is GLYCOGENIN?
- A glycosyltransferase made of 2 identical subunits that each catalyze the addition of 8 glucosyl units to the other subunit
- Forms short α-1,4-glucose polymers covalently attached to the phenolic hydroxyl group of a specific tyrosine residue in each glycogenin subunit
Why must branching occur in glycogen synthesis?
Because it
- increases the solubility of glycogen (will better absorb into cells)
- increases rate of glycogen synthesis/degradation
How accurate is DNA replication?
- Semiconservative model
- Less than 1 erroneous base pair per 3 x 10^9 base pair (bp) (i.e. pretty fucking accurate)
What controls the topological state (supercoiling) of DNA?
- Topoisomerase I
2. Topoisomerase II
What is the function of Topoisomerase I?
-reversibly unwind DNA double helix
How: via cutting and resealing a single strand
-thermodynamically driven
What is the function of Topoisomerase II?
-create negative supercoils
How: via cutting the 2 strands of double helix DNA and passing a dif DNA double helix through the break and then resealing break
-driven by ATP hydrolysis, contrathermic
What is the purpose of supercoiling and how many types are there?
Purpose: Make DNA more compact
Negative Supercoiling: makes strand separation easier
Positive Supercoiling: make strand separation more difficult
What are the functions of DNA POLYMERASE in DNA replication?
- Catalyze 5’->3’ polymerization (adding on nucleotide bases)
- Edit mistakes in base pairs via 3’->5’ exonuclease activity (proofreading)
- Promote the excision of the RNA primer segments in the lagging strand via 5’->3’ exonuclease activity
What are the 2 ways that the new DNA strands are assembled?
- Leading Strand
2. Lagging Strand
How is the LEADING STRAND assembled in DNA replication?
-Nucleotide bases add onto the template strand in the 5’->3’ direction
How: Hydrolysis of pyrophosphate drives 3’-OH end of growing chain to nucleophilic attack a dNTP (nucleotide base)
How is the LAGGING STRAND assembled in DNA replication?
- Primase synthesizes RNA primers
- RNA primers initiate addition of short segments of complimentary RNA
- Each segment continues to extend until it runs into another segment (Okasaki fragment)
- DNA replaces complimentary RNA segments
- DNA polymerase cuts out nick (space) via nick translation until the 2 DNA fragments meet
- DNA ligase seals the nick
What is the KLENOW FRAGMENT?
Large protein fragment made by enzymatic cleavage of DNA polymerase by a proteolysis (occurs in E.coli)
- despite being cleaved, it still retains its 5’->3’ polymerase and 3’->5’ exonuclease activity
- it DOES LOSE its 5’->3’ exonuclease activity
Polymerase active site=in cleft (palm) lined with positively charged residues
Name 2 other characteristics that contribute to the accuracy of DNA replication.
- Hydrogen bonding interactions AND fit between AT and GC base pairs
- Hydrogen bonding at the minor groove by DNA polymerases with the base pairs
How can shape complementarity of base pairs interfere with the accuracy of DNA replication?
- Other molecules can have the same structural shape as base pairs but not be capable of hydrogen bonding
- So, even if the correct base pair matches up, they will lack the ability to hydrogen bond to its pair
How do you unwind DNA?
-Use helicase and ATP hydrolysis
What is a PRIMOSOME?
- Multi protein complex that generates new RNA primers as it move along the lagging strand
- Propelled in 5’->3’ direction by PriA- and PriB-catalyzed ATP hydrolysis
How possible: doesn’t require a primer so it can produce a short RNA oligomer (Okasaki Fragment)
What is a REPLISOME?
- Multiprotein particle containing 2 pol III holoenzymes
- Catalyzes both leading and lagging strand synthesis
What is the function of DNA POLYMERASE III? Its subunits? β-subunit of pol III in DNA replication?
Function: guide replisome along the strand growing complimentary to the lagging strand
- β-subunit: Loosely clamps onto the newly formed end of duplex DNA (or RNA primer DNA duplex) and acts as a sliding clamp that guides the replisome along the growing strand
- γ-subunit: (clamp loader) opens the clamp and moves it to the next initiation site on the lagging strand in an ATP dependent manner
How is DNA replication related to the cell cycle?
-Replication must occur only ONCE during each “cell cycle”–occurs in S-phase
Cell Cycle: S phase: synthesis G2 phase: rest M phase: mitosis G1 phase: rest
What do you call a site/origin of DNA replication in a) humans and b) yeast?
Humans: REPLICON
-there are hundreds of these in the human body
Yeast: AUTONOMOUSLY REPLICATING SEQUENCES (ARS)
What are AUTONOMOUSLY REPLICATING SEQUENCES (ARS)?
Docking sites for origin of replication complexes (ORC) which recruit other proteins
What is the function of LICENSING FACTORS? (DNA replication)
- Allow formation of initiation complex
- Once it has established the initiation complex, they’re marked for destruction by UBIQUITINATION and destroyed by PROTEOSOME via PROTEASOMAL DIGESTION
- destruction ensures that each replicon is replicated only once
What are the steps of initiating DNA replication (in eukaryotes)?
- Helicase unwinds duplex DNA
- Replication protein A coats the single strands and prevents annealing
- After ~20 bases are added to primer, polymerase switching occurs
- Combination of DNA polymerase δ, protein replication factor C (PRC), and PCNA are subbed in for replication protein A
- now elongation process begins
What is POLYMERASE SWITCHING?
The polymerase currently used is switched out for another (or combination of others) that are more efficient at DNA replication
Why: because DNA replication has a limited period (S phase) that it can be done in
What are TELOMERASES?
Enzymes that assemble the ends (TELOMERES) of eukaryotic chromosomes; it has its own RNA template (so doesn’t need a primer)
Why Them: DNA polymerase can’t assemble end b/c it needs a primer and if you were to remove the primer on the lagging strand, it would leave an incomplete 5’ end
How: forms looping structures of a repeating hexanucleotide sequence (AGGGTT) stabilized by telomere-binding protein
What are the 4 ways DNA can be damages?
- Alkylation of N7 in purines->susceptible to being hydrolyzed -> will leave a gap in sequence
- Oxidation of guanine -> 8-oxoguanine -> can pair up with adenine ->mismatch ->mutation
- Hydrolysis (deamination) of adenine -> hypoanthine ->can pair up with cytosine-> mismatch->mutation
- Photodimerization of pyrimidines by UV irradiation -> forms photodimers-> block replication->photolase promotes photocleavage
Describe the DNA SEQUENCE of the protein huntingtin of someone with Huntington’s disease. How could this occur?
Contains 6-20 consecutive glutamine (trinucleotide=CAG) repeats sometimes even more
-Extended repeats make it more likely that the protein will aggregate
How possible: Extra copies of the repeat can be inserted due to the formation of loop structures in the daughter strand
What are the 4 ways that damaged DNA can be repaired? Give examples of enzymes that do these.
- Direct reversal of base modifications
-ex. DNA photolyases, Methyltransferases - Nucleotide excision repair (NER)
-ex. UvrABC endonuclease, DNA glycosylases,
AP endonuclease, uricil DNA glycosylase - Recombination repair
-ex. done by the duplex DNA - Transposition
-ex. transposons/transposable elements
