interrelation of catabolism/anabolism of proteins, carbs, and lipids
all of these paths intertwine and affect each other and they all seem to converge on acetyl CoA for catabolic, or the start at acetyl CoA and branch out from there
catabolism of carbohydrates; exergonic; broken down into energy investment phase and energy generation phase; 10 steps, 3 of which are irreversible; all rxn occur in the cytosol; gross products are 2 pyruvate, 4 ATP and 2 NADH
energy investment phase
hexokinase, phosphohexose isomerase, phosphofructokinase- 1(PFK-1), aldolase, and triose phosphate isomerase
energy generation phase
glyceraldehyde 3 phosphate dehydrogenase (GAPD), phospho-glycerate kinase, phospho-glycerate mutase, enolase, and pyruvate kinase (all 5 of these rxns happen 2x/glucose molecule)
energy investment stage 1: hexokinase is a kinase
kinase: large family of enxymes that catalyze phosphoryl group transfers; these attach Pi to other molecules that is usually, but not always, taken from ATP
energy investment stage 1: hexokinase-- what it does
OH on glucose acts as nucleophile in enzyme active site and attacks phosphate on ATP and then Pi adds where the OH group was and glucose becomes G6P
energy investment stage 1: hexokinase needs to use ATP to work
ΔG'° of adding Pi to glucose is +13.8 and ΔG'° of hydrolysis of ATP is -30.5 so new ΔG'° for the coupled rxn is -16.7, but this is at standard conditions, not in the body. w/o ATP glucose dominates in the cell but with ATP products are favored and G6P dominates
energy investment stage 1: hexokinase-- uses ATP for 2 reasons
1)some of the ΔG'° from ATP hydrolysis will be stored in G6P. 2)use of ATP will drive rxn away from its equilibrium; "far from equilibrium" standpoint-- b/c w/o ATP rxn is endergonic and non-spontaneous and would never occur and we need to production of G6P to be favorable in order for glycolysis to occur
energy investment stage 1: hexokinase-- irreversible reaction
rxn with a single thermodynamically favorable direction at conditions prevailing in the cell; rxn will only go one way and can't go back and thus these irreversible rxns set the direction of the path; aka "far from equilibrium" rxn; every metabolic pathway will have at least one irreversible rxn, most have >1
energy investment phase 1: hexokinase-- ΔG'° vs ΔG
ΔG'° is -16.7 while ΔG is -33.5 so actual starting conditions produce a rxn thats more exergonic and can do more work than standard conditions
energy investment stage 1: hexokinase-- given ΔG'°/ΔG values, the free E minimum of the free E landscape should be to the L or R of 1:1?
R b/c if you start at 1:1 and ΔG'° is negative then this rxn will proceed in the direction of products, its slope will be negative, and its equilibrium (Keq) lies on the product side
energy investment stage 1: hexokinase-- given these ΔG'°/ΔG values, Q should be on the L or R of 1:1?
number of ΔG says how far from equilibrium the rxn is at actual conditions and the sign says in what direction. negative means that equilibrium is on the side of products and the magnitude says that at actual conditions the rxn lies further away from equilibrium than at standard conditions so Q is on the L of 1:1
energy investment stage 1: hexokinase-- why is Q so small
b/ reactants are glucose and ATP and typically when ATP is used as a substrate the Q value is really small b/c the cell keeps very high [ATP] inside the cells which makes the Q value drop ([reactants]>>[products])
energy investment stage 1: hexokinase-- this rxn locks glucose in the cell b/c G6P is a phosphorylated intermediate (high energy compound)
G6P gets locked in the cell b/c GLUT transporters has very high Km's for G6P and thus won't bind it an db/c these GLUT transporters are the only things that glucose can use to get out, ti's stuck till the phosphate is removed-- important in liver.
enzymes that remove inorganic phosphate from molecules-- G6P is dephosphorylated by G6P phosphatase which only resides in the liver b/c liver is important in regulating blood glucose and not in other cells b/c if it was it would release glucose from the cells before glycolysis could finish
energy investment phase 1: hexokinase-- exhibits induced fit via binding of glucose
ATP binds to hexokinase and nothing happens, then glucose binds and hexokinase shuts forming its active site. glucose acts as a nucleophile and hydrolyzes ATP to ADP and takes its Pi to form G6P and rxn is complete. ONLY thing that can induce this conformational change is glucose which is important b/c if ATP could then ATP could get hydrolyzed w/o glucose in there and all that E would go to waste
substrate induces a conformational change in the enzyme active site-- site is inactive before change, and active once change occurs
energy investment stage 2: phosphohexose isomerase-- changing G6P into its isomer
isomerases carry out isomerization which changes a molecule into one of its isomers-- here G6P is changed into F6P
energy investment stage 2: phosphohexose isomerase-- near equilibrium/ reversible rxn
a rxn that is at or near equilibrium and as a result the thermodynamically favored direction is in the opposite direction of an applied stress (le chatlier's principle)-- reversible rxn; ΔG will ALWAYS = 0 for reversible/near equilibrium rxns
energy investment stage 2: phosphohexose isomerase-- ΔG'° vs. ΔG
ΔG'° = 1.7 so slightly endergonic at standard conditions but ΔG = 0 indicating that at conditions prevailing in the cell this rxn is at equilibrium and thus Q - Keq and ΔG = 0 b/c there's no driving force to reach equilibrium b/c you are already there at actual conditions
energy investment stage 3: PFK-1-- similar to hexokinase
kinase that takes Pi off ATP and adds it to F6P making it F16BP, it is an irreversible rxn, ΔG is more exergonic and farther from equilibrium than ΔG'° b/c ATP is a reactant again so Q is very small
energy investment stage 3: PFK-1-- committed step
at all steps before PFK there are branch points out of glycolysis that the diff metabolic intermediates could go down but once an intermediate gets to PFK there are no branching off points until they become pyruvate and thus these carbons are committed to seeing glycolysis through. this is what makes PFK such an important regulatory step b/c if it shuts off it essentially shuts down glycolysis and when turn on it can ramp up pyruvate production
energy investment stage 4: aldolase-- splitter
splits F16BP into DHAP and GAP and now the one glucose molecule is 2 molecules so after step 5 all rxns will be done twice per one glucose molecule. reversible reaction.
energy investment stage 4: aldolase-- ΔG'° and Q
ΔG = 0 thus Q = Keq and the rxn is reversible and ΔG'° = +23.8 so this rxn is very endergonic in standard conditions so Keq will lie on the far left b/c reactants are favored at equilibrium so Keq and Q are very small
energy investment stage 4: aldolase-- what keeps Q so small?
this enzyme makes DHAP and GAP and the enzymes of the following 2 steps are very efficient and clear DHAP and GAP out of the cell so fast that they never have time to accumulate so [products] of this rxn never increases so Q stays small b/c [reactants]>>[products] in the cell
energy investment stage 5: triose phosphate isomerase (TIM)-- conversion of DHAP to GAP
DHAP and GAP are isomers so TIM can convert DHAP to GAP and once this happens there will be 2 identical GAP molecules going through the rest of glycolysis so everything in rxns 6-10 must be doubled. last rxn of the energy investment stage. reversible rxn.
energy investment stage 5: TIM-- efficiency
enediol intermediate is formed in the course of this rxn and this is a HIGHLY reactive intermediate so it is converted to product very quickly partially explaining why TIM is so efficient
energy investment stage 5: TIM-- induced fit
in order for TIM to utilize the efficiency of its intermediate it has to be able to keep it in the active site so it doesn't escape and react w/ other things outside of the enzyme. when substrate bonds a loop changes its conformation and captures the substrate and hold it in place in the active site ensuring the intermediate reacts where it needs to. this conformational change plus the reactivity of the intermediate explains TIM's efficiency
energy investment stage review
5 steps w/ 5 enzymes, 2 irreversible 3 reverisble, 2 ATP hydrolyzed, energy conserved in the 2 GAP from glucose and from phosphorylation from ATP
energy generation stage 6: GAPDH-- dehydrogenase rxn
takes hydride anion w/ 2e- from GAP and H+ from a Pi and reduce NAD+ to NADH + H+ and creates 13BPG-- GAP is oxidized and phosphorylated (acyl phosphate bone). reversible rxn. HIGHLY efficient enzyme.
energy generation stage 6: GAPDH-- 2 steps of rxn
1)oxidation of GAP and reduction of NAD+ w/ ΔG = -43. 2)production of acyl-phosphate bond w/ ΔG = +49. problem-- b/c of huge drop in G in first rxn then the second rxn is very endgergonic and almost impossible so GAPDH byspasses this using thioesters
energy generation stage 6: GAPDH-- rxn mechanism steps 1 and 2
1)cys 149 is deprotonated and nucleophilically attacks GAP forming thiohemiacetal intermediate 2) redox step-- NAD+ takes H off intermediate and His 176 removes another H from intermediate causing a rearrangement of e- creating a thioester bond that is very high in energy and can thus store all of the energy released from the redox step
energy generation stage 6: GAPD-- rxn mechanism remaining steps
3)Pi can now nucleophilically attack the thioester intermediate and break the thioester bond providing a release of energy to pay for the +49 required to attach the Pi making GAP 13BPG
energy generation stage 6: GAPDH-- roles of cys 149 and his 176
at physiological pH cys can't be deprotonated but in the active site pH is lowered enough for this to be possible so it can attack and attach to GAP and then His can act as a base taking another H from GAP after NAD+ takes one and this ultimately results in the high energy thioester bond bridging the energy gap b/c GAP and 13BPG
energy generation stage 7: phosphoglycerate kinase-- substrate level phosphorylation
ATP production in which G derived from the hydrolysis of a "high energy compound" higher in ΔG than ATP is utilized to phosphorylate ADP to make ATP thus this step produces a total of 2 ATP and 2 3PG
energy generation stage 7: phosphoglycerate kinase-- Q and ΔG'°
ΔG'° is very negative so Keq<<1:1 but ΔG = 0 so in the cell this rxn is reversible and Q = Keq. this is b/c whenever ATP is the product of a rxn then Q is going to be very large meaning that products are highly favored b/c the cell has so much ATP in it
energy generation stage 8: phosphoglycerate mutase
makes 3PG 2PG by rearranging where the Pi is attached just b/c at the new point the bond as a higher energy than at the old point. reversible rxn.
energy generation stage 9: enolase
turns 2PG into PEP which is the highest high energy compounds w/ a ΔG'° of hydrolysis =~ -61. uses metal ion catalysis.
energy generation stage 10: pyruvate kinase
uses substrate level phosphorylation to make 2 ATP and 2 pyruvate molecules. b/c so much more G released in hydrolyzing PEP than can be stored in ATP the ΔG of this is -17 so irreversible and Q > 1:1 while Keq is >> 1:1 b/c ΔG'° is -31.4. Q is so large again b/c ATP is a product.
resupplying the cytosol with NAD+
2NADH made in glycolysis can be used in mitochondria to make ATP via donating their e- but after it drops off e- it must become NAD+ again to replenish the cytosol's supply. 3 ways this is done.
mitochondrial membrane shuttles
no way for NAD/NADH to cross into mitochondrial matrix b/c no transporter in the membrane so shuttles must exist to move e- across the matrix membrane so the e- can be used in e- transport chain. 2 aerobic shuttles and 1 anaerobic
aerobic conditions: malate-aspartate shuttle
OAA in cytosol is reduced by NADH to malate and NADH is oxidized to NAD+ resupplying the cytosol. malate moves into mitochondria and is oxidized by NAD+ back to OAA. glutamate gives OAA an amino group making it aspartate and glutamate becomes aKGA that moves out of mitochondira where it takes amino group from aspartate turning that into OAA and aKGA becomes glutamate and everything can start over
aerobic conditions: glycerol-3-phosphate shuttle
cGPD oxidizes NADH to NAD+ resupplying cytosol and reduces DHAP to glyerol-3-phosphate which is oxidized by mGPD back to DHAP and FAD is reduced to FADH2 and then FADH2 gives e- to Q reducing it to QH2 and now e- are in mitochonria. not as efficient of malate-aspartate shuttle. FAD/FADH2/mGPD are all one complex embedded in matrix membrane
O2 is no longer available and cellular respiration shuts down so mitochondria shuts down so ATP production in mitochondria shuts down and so do its 2 shuttles. ATP demand shoots up and the only thing making it is glycolysis so now super important that NAD+ gets replenished
anaerobic conditions: lactate dehydrogenase
reduces pyruvate to lactate and oxidizes NADH to NAD+. occurs in cytosol. exergonic and irreversible. this process is called lactate fermentation.
glycolysis in summary
Keq = 1014-- highly favors pyruvate. ΔG = -90 so very exergonic. efficiency = 44% at standard and 53% in cell so better in cell but still not great.
gluconeogenesis-- why do it?
done in the liver b/c liver is key in maintaining blood glucose levels. not only that but glucose is the only form of energy that the brain can utilize so it is CRUCIAL that the liver can make glucose, and has lots of different ways to make it in case CHOs arent readily available, to keep the brain supplied with energy
gluconeogenesis is NOT the reverse of glycolysis
if it was ΔG would be +90 but ΔG is -16 so something has to be different. different b/c glycolysis has irreversible steps thus GNG must use different enzymes and different mechanisms to bypass these steps so it is NOT the exact reverse of glycolysis. also, it requires 6 ATP and 2 NADH in order to work. GNG steps bypassing irreversible step of glycolysis will also be irreversible
irreversible glycolysis rxns and GNG steps to bypass them
pyruvate kinase-- bypassed by PEP carboxylase and pyruvate carboxylase. PFK-- bypassed by F16BPase. hexokinase-- bypassed by G6Pase
irreversible rxn of glycolysis 1: pyruvate kinase
takes Pi from PEP making ATP and pyruvate. is highly exergonic and irreversible. opposing GNG reaction uses 2 different enzymes to bypass this step
bypass rxn of GNG 1: part 1-- pyruvate carboxylase
hydrolyzes ATP to add a carbon from bicarbonate to pyruvate to convert it to OAA. occurs in the mitochondria. occurs twice b/c 2 moles of pyruvate made/glucose molecule so really 2 ATP hydrolyzed
bypass rxn of GNG 1: part 2--PEP carboxykinase (PEPCK)
decarboxylates OAA and releases carbon as carbon dioxide and then hydrolyzes GTP to GDP to phosphorylate OAA making PEP. b/c this rxn has to run twice 2 GTP are hydrolyzed in this process.
rxns 2 - 6 of GNG
these were all reversible in glycolysis so these steps in GNG truly are the opposite of the ones in glycolysis. once PEPCK makes PEP, it will put pressure on enolase rxn to run in the reverse and create more reactant which is the product of the rxn before it so it causes that rxn to run in the reverse as well, and on and on
phosphoglycerate kinase rxn in GNG
in glycolysis this produces ATP therefore in GNG it must expend ATP and b/c it happens twice it will use 2 ATP
GAPDH rxn in GNG
makes NADH in glycolysis so in GNG it must expend NADH making NAD+ so a total of 2 NADH oxidized per GNG cycle-- means that in GNG it is the NADH that must be replenished in the cytosol as opposed to NAD+ in glycolysis
aldolase rxn in GNG
in glycolysis this is the rxn that splits the molecules so here it will put them back together and for the rest of GNG there will no longer be doubles of everything
removes Pi from F16BP making it F6P. even though in glycolysis PFK hydrolyzes an ATP to add Pi, in GNG an ATP is no produced b/c this is not the true opposite of the PFK rxn. important regulatory checkpoint of GNG.
bypass rxn 3 of GNG: step 10-- G6Pase (opposes hexokinase)
removes the last phosphate making glucose that can be shipped out of the cell via glut transporters. even though hexokinase hydrolyzes an ATP, this rxn does not produce one. G6Pase only found in certain tissues ie liver, but not muscle
GNG in skeletal muscle
skeletal muscles don't have G6Pase thus can't make G6P into glucose so the end product of GNG here is G6P which can't be excreted from the cell b/c of Pi, but it can be stored as glycogen
making a single glucose molecule requires 4ATP, 2GTP, and 2NADH
replenishing the supply of NADH in the cytosol from GNG
as the rate of GNG increases, then the supply of NADH decreases so there must be a way to replenish it in the cytosol BUT not NADH transporter to mitochondria where its e- are going to it needs a shuttle
Malate Shuttle/Shuffle-- if pyruvate comes from anything other than lactate
pyruvate crosses into matrix where pyruvate carboxylase makes it into OAA and then MDH converts OAA into Malate by oxidizing an NADH to NAD+ and now Malate can move into cytosol where it reacts with MDH again to become OAA this time reducing NAD+ to NADH replenishing NADH supply in the cytosol. now OAA can interact with PEPCK to make PEP for GNG
Malate Shuttle/Shuffle-- if lactate is the source of pyruvate
LDH will oxidize lactate to pyruvate by reducing NAD+ to NADH replenishing NADH in the first step. pyruvate then goes into matrix and reacts with pyruvate carboxylase to make OAA which reacts with PEPCK to make PEP for GNG which can then move back out to cytosol
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