biochem lecture 5 pt 1 Flashcards
common intermediate example
acetyl CoA
what do we have a often in catabolic pathways
convergence of diff catabolic processes that converge at a common intermediate
what does common intermediate represent
common currency that can be used to make ATP
what diff pathways will converge at level of acetyl CoA
glycolysis, oxidation of fatty acids, degradation of AAs
basically what does this mean
divergent energy sources, but converge thru formation of common intermediate acetyl CoA
why is acetyl coA important
these two carbon fragments can all be used or oxidized via the TCA cycle regardless of whether they come from glucose or fatty acids etc
what is importance of acetyl CoA
reducing power; can extract e- from Carbons, and transfer to electron carriers like NAD and FADH
where does most of reducing power in cell respiration come from
TCA cycle
what are three stages
partial oxidation via glycolysis of glucose into pyruvate, pyruvate into acetyl CoA by PDH complex, and other 2 Cs from pyruvate will be oxidized in oxidative decarboxylation step
what happens to pyruvate in TCA cycle
complete oxidation of 3 Cs
what happens to all 3 carbons in pyruvate in TCA cycle
released as waste in form of CO2
describe TCA
amphibolic
amphibolic
plays a role in catabolism and anabolism
what is TCA a central pathway for
recovering E from several metabolic fuels
what can TCA intermediates serve as
precursors for biosynthetic pathways
what is advantage of a cycle
you can have intermediates that are generated, and siphoned off into other pathways
why do we have cycles instead of linear pathways
intermediates can be directed out of cycle to other pathways, or can have things going into pathways to continue cycle
anapleurotic reactions
rxns that replenish intermediates depleted by other rxns
what can some intermediates in TCA cycle be used for
biosynthesis of fats, amino acids, components of heme or porphyrin ring structure (succinyl CoA)
what do cytochromes found in ETC have
protoporphyrin ring structures, heme-like structures
where are some gluconeogenesis precursors derived from
TCA cycle (like oxaloacetate)
what is TCA cycle important for
both catabolic and anabolic processes
how are intermediates maintained
anaplerotic rxns
anapleurotic reactions
rxns that replenish intermediates that have been depleted by other reactions, maintain levels
why is replenishment reactions important
if we have lowering of oxaloacetate (end product of TCA), it reduces level of flow/flux thru pathway, inhibits overall flow
what is having replenishment rxns necessary for
maintaining flux/flow thru the cycle
important precursors in gluconeogenesis
malate –> Oxaloacetate –> glucose
important precursors in lipid biosynthesis
citrate –> oxaloacetate + acetyl coA –> lipids
precursors in porphyrin biosynthesis
succinyl CoA
what pathways use TCA cycle intermediates
gluconeogenesis, lipid biosynthesis, AA biosynthesis, porphyrin biosynthesis
what is succinyl coA important for
heme production
where does TCA cycle take place
mitochondrial matrix
where does glycolysis take place
cytosol/cytoplasm
describe mitochondria
double membrane organelle, has microcomparments
describe outer membrane of mitochondria
permeable to small things (less than 5 kD)
describe inner membrane of mitochondria
impermeable; only permeable to O2, H2O, CO2
what does other stuff require in mitochondria
transport proteins
describe structure of mitochondria
folded inner mitochondrial membranes, cristae
what is importance of cristae or folded membrane
can increase internal surface area
what happens in inner mitochondrial membrane
its where components of ETC and ATP synthase reside
what does increased SA mean
you can localize more copies of ETC and ATP synthesizing components
endosymbiosis theory
mitochondria arose from symbiotic relationship b/w bacteria and eukaryotic cell (bacteria had cushy environment, cell could harvest its ATP)
which is more permeable, inner or outer membrane
outer membrane
why is relative impermeability of inner membrane important
for when we talk about ATP synthesis, and establishing a proton gradient across inner membrane
what is required for shuttling things into and out of mitochondria
transport mechanisms
where is glycolysis
cytoplasm
where is TCA cycle and ATP synthesis
mitochondria
so what happens if pyruvates generated in glycolysis need to undergo complete oxidation in TCA cycle
need to be transported to mitochondria
big picture of TCA cycle; first step
pyruvate from glycolysis is split to acetyl CoA
what does first overall big picture step (pyruvate –> acetyl CoA ) generate
1 NADH, 1 CO2
what is second big picture step
acetyl Coa + oxaloacetate –> citrate
what happens to citrate
enters cycle
what is 3rd big picture step
one 2 carbon acetyl group of citrate is oxidized
how many steps in it
8 steps; 2 CO2, 3 NADH, 1 FADH2, 1 GTP/ATP
what is generated at the end of TCA cycle
oxaloacetate
what do pyruvates derived from glycolysis undergo
preparative step or activation
does pyruvate itself enter TCA cycle?
nope, needs to be converted to acetyl CoA
how are pyruvates converted to aectyl coa
pyruvate dehydrogenase complex
what enters the TCA cycle
acetyl CoA
what do we generate in this prep step
1 NADH, first CO2 thats produced (from converting pyruvate to acetyl coa)
what are nadh/fadh2
money in the bank, reduced electron carriers that can be cashed in the ETC and used to generate ATP
what happens after acetyl coa is amde
2 carbon fragments joined to coenzyme A (acetyl CoA) combine w/ oxaloacetate –> citrate
what is oxaloacetate
end product of TCA cycle
what is starting point of TCA
citrate
what kind of rxn involved in formation of citrate
condensation reaction (where you combine 2 carbon acetyl units from acetyl CoA w/ oxaloacetate)
what happens for each acetyl CoA that enters cycle
8 rxns overall
how many CO2s do we generate
2 CO2s per turn of the cycle
how many oxidative decarboxylation steps
2 oxidative decarboxylation steps
how many carbons from acetyl CoA will be completely oxidized
w carbons
what do we generate per cycle
3 NADH, 1 FADH2, 1 GTP/ATP
what is important for cycle to continue
regeneration of oxaloacetate
how many pyruvates per glucose do we generate in glycolysis
2
so how many acetyl CoAs
2; (2*2 = 4 carbon acetyl units)
how many turns of cycle for complete oxidation of a single glucose molecule
2 turns
so how much do we produce per glucose
6 NADHs, 2 FADH2s, 2 GTP/ATPs, 4 CO2s per glucose, every 2 turns of cycle
what is prep step
production of acetyl coA from pyruvate
why is prep step necessary
allows for oxidation of carbons in TC cycle
what does coenzyme A have
reactive thiol group
what happens when thiol group is linked to two carbon acetyl units from pyruvate
thioester linkage
what is acetyl coa an example of
high energy intermediate
what happens when thioester bond in acetyl coA is cleaved in first step of TCA cycle
2 carbon acetyl unit is gonna be transferred to oxaloacetate (to form citrate), and free E is released
what is free E released from cleavage of thioster bond used for
to form citrate from acetyl coa. + oxaloacetate
basically what does hydrolysis of thioester bond of high E intermediate acetyl coA used for
provides E needed to carry out first step in TCA cycle
basically what is preparation
generation of acetyl CoA from pyruvate
what does coenzyme A receive
2 carbons (in form of acetyl group)
where does coenzyme A receive 2 Cs from
pyruvate
what linkage is formed in prep step
thioester linkage (acetyl CoA)
what energy complex takes pyruvate from glycolysis to produce acetyl CoA
pyruvate dehydrogenase complex
what is pyruvate dehydrogenase complex
massive complex, multi sub unit, many components
what coenzymes does pyruvate dehydrogenase complex utilize
coenzyme A, NAD, FAD, TPP, lipoic acid. etc
what else is generated in prep step (pyruvate –> acetyl CoA)
NADH and CO2 (first co2 produced)
how many rxns in prep phase
5 sequential rxns; oxidative decarboxylation
what is oxidative decarboxylation
where you generate CO2 thru a series of redox rxns that take place in this enzyme complex
what does pyruvate dehydrogenase have
multiple copies of these 3 enzymes
what 3 enzyme/subunits are in pyruvate dehydrogenase complex
pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), dihydrolipoyl dehydrogenase (E3)
E1
pyruvate dehydrogenase
E2
dihydrolipoyl transacetylase
E3
dihydrolipoyl dehydrogenase
how many copies of E1, E2, E3
multipleeee
what is PDH complex an example of
substrate channeling
what is substrate channeling
series of sequential rxns that hand off intermediates to each other
what happens to the intermediates in substrate channeling
we never lose them, they are effectively trapped
how are the intermediates trapped
thru formation of covalent intermediates within enzyme complex
what does substrate channeling allow for
more efficient catalysis; cuz intermediates are tethered to coenzymes in enzyme complex
what is step 1
E1 carries out oxidative decarboxylation step
what is released in step 1
CO2; 1 carbon from carboxyl group of pyruvate is released
what happens to the remaining 2 carbon fragment in pyruvate
transferred to a coenzyme TPPP
what is TPP formed
hydroxyethyl TPP
what is hydroxyethyl TPP tethered to
TPP coenzyme
what does E1 complex do
hands off acetate unit to next coenzyme (lipoic acid)
what is the next coenzyme
lipoic acid
what is lipoic acid here called
lipoyllysine
why lipoyllysine
cuz its tethered to a specific lysine in E2 complex
where does 2 carbon fragment from hydroxyethyl TPP go
transferred to acyl lipoyllysine
what is lipoic acid basically like
a long arm, like arm of crane as it rotates
where does lipoic acid rotate
in E2 subunit
what is acetate tethered to
acetate is tethered to partially oxidized lipoyllysine
what does the tethered acetate undergo
partial reduction; one of 2 sulfurs is reduced to thiol
when is this partial reduction happening
in process of transferring acetate from TPP from E1 to lipoic acid in E2
what is step 3
E2 subunit is gonna transfer acetate group to a molecule of coenzyme A
what mediates the actual synthesis of acetyl CoA
E2
what does E2 do
generates acetyl CoA which can go into TCA cycle
so are we done after E2?
nope
why aren’t we done
once we’ve transferred this 2 carbon acetate unit to generate acetyl CoA in step 3, we are left w/ a fully reduced form of lipoyllysine
what is CoA-SH
reduced form of coenzyme A
what is lipoyllysine
cofactor that’s attached to a lysine within E2 polypeptide
why is having the two thiol/SH groups here a problem
cuz in order for E2 to accept another 2 carbon fragment from hydroxyethyl TPP of E1, it needs to be in fully oxidized form
what is fully oxidized form of lipoyllysine
disulfide bridge form
what does lipoyllysine start out as
fully oxidized form; disulfide bridge
what are steps 4 and 5 important in
regeneration of fully oxidized lipoyllysine of E2
where does E3 come into play
in regenerating oxidized lipoyllysine
what would happen without reoxidation of lipoyllysine
E1 can’t transfer the two carbon fragment to lipoyllysine to generate another acetyl CoA; everything would stop
what does E3 do in order to generate oxidized lipoyllysine from the reduced form here
sets up sequential redox reactions in steps 4 and 5
what does step 4 involve
FAD: reduced lipoyllysine is gonna transfer 2 Hs (2 electrons) to FAD
why is step 4 a win-win
we regenreate oxidized lipoyllysine (needed to keep stuff going) and also generate some reducing power in form of FADH/FADH2
what happens if we generate reduced FAd/FADH2
we need a pool of oxidized FAD to keep E3’s regeneration step going
what is step 5
a second redox step in which FADH2 is reoxidized
what else is produced in step 5
we generate reduced NAD so NADH
why is E3 subunit important
cuz it regenerates oxidized lipoyllysine and oxidized FAD (so that E2 can carry out regeneration)
how do we reoxidize FADH2 to FAD
by setting it up w/ another redox rxn involving NAD (NADH)
why is it not enough to have first 3 steps
cuz its not enough to just produce acetyl CoA; E2 subunit needs to reset itself
why is E3 so important
cuz its gonna regenerate oxidized form of lipoic acid to keep things going
what is E1
pyruvate dehydrogenase
what does E1 do
forms hydroxyethyl TPP intermediate
basically what does E1 do (how does it form hydroxyethyl TPP intermediate)
uses TPP as a cofactor, attacks the carbonyl C2 of pyruvate (releases CO2), and TPP is bound to hydroxyethyl group –> hydroxyethyl TPP
what is lipoic acid
cofactor that is covalently tethered to specific lysine residue of E2
what for form of lipoic acid is involved
oxidized form of lipoic acid
what is lipoic acid involved in
in transfer of acetate (2 carbon) units from hydroxyethyl TPP intermediate to lipoic acid
what form of lipoic acid is gonna be used in synthesis of acetyl CoA
hemi/partially reduced / acetylated form
what form of lipoic acid are we left with
reduced form
what has to happen to the reduced form of lipoic acid
has to be re-oxidized (by steps 4 and 5, E3 subunit)
basically what is E3, steps 4 and 5 all about
getting lipoyllysine back to fully oxidized form so it can accept another acetate unit from hydroxyethyl TPP intermediate from E1
what’s involved w/ E2
lipoamide side chain extends to E1. transfers hydroxyethyl from TPP to dihydrolipoamide. partial reduction creates acetyl group, second reduction transfers acetyl group to CoA
how is acetyl CoA generated
coenzyme A receives 2 Cs from pyruvate in form of acetyl group
what linkage does acetyl CoA have
high E thioester linkage
what do we need to regenerate after we generate acetyl CoA
oxidized lipoic acid to keep things going, so it can accept more pyruvate
what does E3 do basically
resets the system
what’s up w/ E3
catalyzes regeneration of disulfide/oxidized form of lipoamide. uses bound cofactor FAD (reduced to FADH2). NAD+ oxidizes FADH2 to regenerate FAD. NAD becomes reduced (NADH_
what happens after E3
PDH enzyme complex is regenerated. NADH is made
what is PDH complex tethered by
lipoamide arm
what happens to intermediates in PDH
intermediates never leave complex
what is stage 2 of TCA
oxidation of acetyl CoA
what is one of major waste products generated in TCA
CO2
how else is acetyl CoA made
fatty acid oxidation, amino acid degradation
how many steps in stage 2
8 steps
what are products of stage 2
3 NADH, 1 FADH2, 1 GTP/ATP (per cycle; multiply by 2 to get per glucose unit)
what are byproducts of stage 2
H2O, CO2
common intermediate acetyl coA
whether it comes from pyruvate (like we discussed), or from oxidation/degradation of fatty acids or AAs, it can be fed into the TCA cycle
first step of TCA
combo of acetyl CoA (2 carbon fragment) w/ oxaloacetate (end product) –> citrate
how many steps in TCA cycle
8 steps
what happens in steps 3 and 4
sequential oxidative carboxylation –> CO2 generated in steps 3 and 4 AND NAD reduced to NADH
how can acetyl CoA be derived rom fatty acids
beta oxidation
what is major source of E
fatty acids
per molecule who gives more E
fatty acids
but what is preferred E source for organsims
glcuose; faster and easily obtained
step 1 of TCA
formation of citrate from acetyl CoA and oxaloacerate
what enzyme for step 1
citrate synthase
what kinda rxn in step 1
condensation
what C of acetyl group is joined to what of OAA
methyl C of acetyl group is attached to carbonyl C of oxaloacetate
what happens to free CoA-SH (reduced); that’s not used in condensation rxn
goes back to PDH complex
what is step 2 of TCA
formation of isocitrate from citrate
what enzyme in step 2
aconitase
describe step 2
citrate –> cis-aconitate –> isocitrate (2 rounds of aconitase)
what intermediate formed in step 2
cis-aconitate C2-C3 double bond intermediate
how many steps in step 2
2 steps
what is step 3
oxidation of isocitrate
what do we get in step 3
isocitrate –> a-ketoglutarate
what kinda rxn is step 3
a 3-step oxidative decarboxylation
what enzyme in step 3
isocitrate dehydrogenase
describe step 3
isocitrate –> oxalosuccinate –> a-ketoglutarate
what does initial oxidation of isocitrate do in step 3
oxalosuccinate intermediate; produces first NADH
what does decarboxylation give in step 3
oxalosuccinate –> alpha-ketoglutarate; CO2 is produced
what ion in enzyme active site stabilizes intermediates in step 3
manganese
what is step 4
oxidation of a-ketoglutarate
what kinda rxn is step 4
another oxidative decarboxylation
what enzyme in step 4
a-ketoglutarate dehydrogenase complex
what is a-ketoglutarate dehydrogenase complex similar to
PDH complex
what happens in step 4
a-ketoglutarate –> succinyl CoA
what else is produced in step 4
Co2, NADH
similarities b/w PDH and enzyme in step 4
same cofactors (TPP, FAD, NAD)
differences b/w PDH and enzyme in step 4
produces succinyl CoA instead of acetyl CoA; AND produces second NADH and Co2
describe energy of succinyl coA
high energy
what is step 5
succinyl CoA –> succinate
what enzyme in step 5
succinyl CoA synthetase
what else is produced in step 5
produces GTP or ATP as well as CoASH
what kinda rxn is step 5
substrate level phosphorylation
what do u need to look for anytime u have substrate level phosphorylation rxn
high E intermediate (succinyl CoA)
what does whether GTP or ATP is produced depend on
S-CoA-synth isozyme
what drives ATP/GTP formation in step 5
free E released in breakage of thioester bond
what is step 6
oxidation of succinate
describe step 6
succinate –> fumarate
what enzyme in step 6
succinate dehydrogenase
what else is generated in step 6
FADH2 (reduced FAD)
where is FADH2 generated
step 6
what is FAD bound to
covalently bound to enzyme along w/ iron-sulfur centers
where do electrons flow in step 6
from FAD - iron/sulfur - ETC
what does this electron flow ultimately lead to
ATP production (ox/phos)
where is succinate dehydrogenase
embedded in inner mitochondrial membrane
what is complex 2 of ETC
succinate dehdyrogenase
what is succinate dehydrogenase
complex 2 of ETC
what is step 7 of eTC
hydratino of fumarate
what enzyme for step 7
fumarase
what happens in step 7
fumarate –> malate
how do we go from fumarate to malate (what is added)
H2O added across fumarate double bond
what kinda fumarate does this work with
only trans, notcis
what is step 8 of TCA
oxidation of malate
what enzyme in step 8
malate dehydrogenase
what happens in step 8
L-Malate –> dehydrogenase
what else is produced in step 8
3rd NADH (malate kicks it out)
what happens w/o oxaloacetate
we can’t keep cycle going
why is malate important
regenerates OAA for another cycle of TCA
where is TCA regulated
3 exergonic steps / rate limiting steps
what 3 steps is TCA regulated
citrate synthase (1st step), isocitrate dehydrogenase, a-ketoglutarate dehdyrogenase
what does it mean if a rxn is exergonic
mostly one directional pathway/step
are exergonic rxns forward or reverse
mainly forward
why are exergonic steps-rate limiting
if you deplete [ ] or have lower levels of substrates, used up rapidly, need to keep replenishing to keep it going
what inhibits/activates the above enzyme
when you have high concentrations of intermediates (citrate, succinyl CoA, NADH), serves as feedback inhibitors of citrate synthase and dehydrogenases
what determines whether the cell is under catabolic mode or anabolic mode [AKA which pathways are operational]
E state of cell
what are indicators of energy state of the cell
specific intermediates and reduced electron carriers
what does high [ ] of intermediates like citrate or succinyl CoA mean
we have a lot of NADH, so reducing power, so high E
what are indicators of low E
lower amounts of reduced e- carriers, more NAD+, more AMP/ADP
what do we have in muscles
calcium
what is calcium
important activator of isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase
what is the point
you can turn pathways on or off at exergonic steps
describe these exergonic steps
unidirectional, favorable steps
what happens if you shut down reversible steps
might block potentially reversible/opposing steps that occur in anabolic processes
how can we most effectively control the amount of flux/flow in a pathway
at these unidirectional, exergonic, rate limiting steps
indicators of high/low E staet
ATP, NADH, acetyl-COA, ADP, NAD+, coA, calcium
what compounds are inhibitory
ATP, NADH, acetyl coA
what compounds are stimulatory
ADP, NAD+, CoA, Calcium
what do these inhibitory compounds serve as
feedback inhibitors of catabolic steps; indicators of high E state
what kinds of compounds are stimulatory
stuff that indicates low E state
key regulatory points in TCA
citrate synthase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase
