TCA and Mitochondria Flashcards
What are some of the key features of the mitochondria?
- Outer membrane is permeable to most molecules under 6kda
- Inner membrane is VERY IMPERMEABLE, contains cardiolipin almost no cholesterol
- allows voltage and proton concentration gradients to be established, generated using ATP
What does mitochondrial DNA do? How does it effect proteins in the mitochondria?
- Mitochondria have own small genome, machinery for transcription and translation
- Encodes a few proteins of the ETC, and tRNAs used in translation of mitochondrial genes, but most come from nuclear DNA and are delivered there
- Reproduce by fission, inherited maternally
Why is mtDNA hypermutable?
- naked, not bound to histones in chromatine structure
- exposed to high concentrations of reactive oxygen species (ROS), damage increases with age
- Genome has limited ability to repair DNA damage
How does mtDNA over come hypermutability?
- Multiple mitochondria in each cell
- Multiple copies of mtDNA in each mitochondria
How are mt diseases heterogenous?
- Normal and mutant DNA can be present in the same person/cell at diff ratios
- Results in a RANGE of phenotypes for diseases caused by mutations in mt genes
- diseases tend to be progressive, get worse with age
What is the role of Pyuvate dehydrogenase?
Links Glycolysis to the TCA cycle, defects in PDH lead to seroius neurological conditions.
Where are glycolysis, PDH and the TCA cycle in a cell?
Cytosol- glycolysis
Mitochondria- PDH and TCA
What protein transports pyruvate into the mitochondria? What happens if it inherits point mutations?
- MPC (MItochondrial pyruvate carrier
- mutations cause lactic acidosis and hyperpyruvatemia
How many subunits does PDH have and what do they do?
4 subunits
E1- pyruvate decarboxylase (TPP)
E2- Transacetylase (lipoate)
E3- Dihydrolipoyl dehydrogenase (FAD,NAD+)
How is PDH regulated?
-Activated: acetyl CoA, NADH
Phhosphorylation of E1 turns off PDH, swicth to FA metabolism
-Deactivated: Pyruvate and ATP, dephosphorylation of E1 turns on PD
What does PDH phosphatase do?
Has reverse effect on PDH kinase and activates E1, activated by Ca
What are the sources of acetyl coA?
Fatty acids, ketone bodies, glucose, pyruvate, ethanol
What is the key rate limiting enzyme in the TCA cycle?
Isocitrate dehydrogenase
What are the two energetically unfavorable (positive G) reactions?
- Citrate to Isocitrate (reverse favorable)
2. Malate to oxaloacetate (“”)
What does succinate dehydrogenase do?
Oxidizes succinate to introduce double cc bond, reducing FAD and creating fumarate
- SD is a component of the ETC, creates proton gradient for generation of ATP
- Tumor supressor gene, causes mutations in enzyme that generates fumerate, increase conc of fumerate which out competes ketogluterate
How many ATP are created during the TCA cycle?
10 ATP
2 carbon acetyl groups and 2 waters, create 2 CO2
What happens to NAD and NADH when energy is consumed?
What effect does this have on oxaloacetate?
What effect does this have on isocitrate?
NAD increases (moves rxn forward), NADH decreases
Oxaloacetate increases
Isocitrate decreases
What happens when energy is abundant?
What effect does this have on malate? Isocitrate? Citrate?
Nad+ decreases, NADH increases
Malate- increase, isocitrate–won’t change much (hump), citrate–increases
What does ethanol metabolism do?
Produces NADH.
In alcohol poisoning massive amount of NADH produced locks up TCA cycle.
What regulates isocitrate dehydrogenase?
Ratio of ADP to ATP in mitochondria. (allosteric)
Activator-ADP, indicates e shortage, increase TCA cyle to make more FAD, more e
Inhibitor-ATP, NADH
How is the TCA cycle amphibolic?
It is catabolic and anabolic
Catabolic- TCA reduces NAD and FAD for generation of ATP in ETC
Anabolic- TCA intermediates are feedstock for biosynthetic pathways
What are TCA intermediates? What type of biosynthesis do they promote?
Malate--> glucose Oxaloacetate--> AA Citrate--> FA alphaketoglutamate --> glutamate--> GABA Odd chain fatty acids--> succinyl coA--> heme biosynthesis
How does the TCA cycle replace intermediates that leave for other pathways?
- Pyruvate carboxylase–converts pyruvate to oxaloacetate
- Pyruvate–From AA or Carbs
- Glutamate- deaminated to make a ketoglutarate
- Branched chain AA converted to propionyl coA, then succinyl CoA
- Direct transamination of aspartate to malate
When exercising, the amount of oxaloacetate can be a limiting factor for the production of E through the TCA cycle. What pathway supplies oxaloacetate to TCA cycle?
Use ATP to “recharge the batteries”
What is myoadenylate deaminase deficiency?
Inherited mutation in gene coding for AMPD1 isoform of AMP deaminase, results in inactive enzyme.
short term–increase in glycolysis and acidosis
long term–myoglobin in urine and muscle cell breakdown
**muscle pain, weakness when exercising, no increase in blood amonia after exercising, no AMPD1 in muscle biopsy
What is the ETC made up of? What are inputs and outputs?
Complex 1,3,4, 5 (atp synthase)
Input NADH get ATP
What is complex 1?
NADH Dehydrogenase–oxidizes NADH and transfers e through molecular wire, pumps 4 protons from matrix to intermembranl space
NADH to FMN2H to FES to CoQ
What is CoQ?
Exists in inner membrane, can accept 1 e than another to make CoQH2 (fully reduced form), is a source of oxidative stress
What happens in complex 3?
Cytochrome bc complex
CoQH2 transfers e from cytochrome B to iron sulfur center than to Cyt C
Used to pump 4H from matrix
What happens at complex 4?
Cytocrome c oxidase
e- go to O2 to make water, cytC pumps 2H
What does the incomplete reduction of oxygen do?
Forms 3 radicals: ROS (reactive oxygen species)
- Hydrogen peroxide
- hydroxyl radical–can also be generated non-enzymatically from superoxide and hydrogen peroxide
- Superoxide dismutase (cytosol Cu-Zn-SOD
mitochondria: MN-SOD), turns superoxide into peroxide, can neutralize peroxide to water, if doesn’t happen, peroxide makes hydroxyl radical–damges proteins, lipids, and goes on to cauesmore damage…why mito diseases are PROGRESSIVE
What do H do in the intermembrane space?
Have potential energy that wants to relieve concentration gradient, so they go through the ATP synthase
Describe the structure of ATP synthase.
F0- 12 C subunits, each have channel for 1 proton
F1- three alpha beta pairs (three catalytic subunits can generate 3 molecules of ATP)
-1 rotation moves 12 H and makes 3 ATP
How is the ETC coupled to the generation of ATP?
- ATP synthase activity stops (ratio of ATP to ADP high) ETC stops
- ETC blocked (hypoxia, no final acceptor for e), inssuficient proton movtive fore to run ATP synthase
What is uncoupling?
Transfer of e- from NADH to O2 without generation of ATP
What are the three types of uncoupling?
- Adaptive thermogenesis: normal response to cold
- chemical uncoupling: toxins transport protons from intermembrane space to matrix
- mechanical uncoupling: damage to membranes equalizes protons concentration between intermembrane space and matrix
What is Adaptive thermogenesis?
Normal response to cold–Allows fat to be utilized for heat independently of ATP consumption
Norepinephrine released, activates lipase which form free FA from triacylglycerol in brown fat cells, proton channel (thermogenia) is also activated.
What is chemical uncoupling?
DNP
Lipid soluble molecules iwth a pka near neutral:
bind H in the intermembrane space where H is high, diffuse across the inner membrane, release a H in matrix where H is low
What is mechanical uncoupling
Due to membrane damage: peroxidation by ROS or mitochondrial swelling due to influx of water
H leaking through holes in membrane make it impossible for ETC to maintain sufficient concentration gradient for ATP synthase to fxn
The inner mitochondrial membrane is only permeable to:
O2, NH3, CO2, Water
all other molecules use transport channels
Where does the energy for transport of molecules across the mitochondrial inner membrane come from?
- Electrochemical gradient (matrix side is negatively charged compared to intermembrane space side)
- pH gradient: H is lower in matrix than in intermembrane space
What are the 3 basic types of transport systems?
- Antiporters- transfer 1 molecule in and another out (atp/adP
- Symporters- transport 2 molecules in or out (pyruvate + H, Pi + H=)
- Uniporters–transport 1 molecule in or out (Calcium)
What is the mitochondrial permeability transition pore (MPTP)?
Caused by hypoxia, large copmlex between ANT,VDAC and other proteins. Causes depolarization of mitochondrial membranes and disrupts protein gradients.
Activation–leads to cell death (apoptosis) or necrosis (nonprogrammed cell death)
