MCB 2000 Chapter 16

what is glycolysis

glucose to pyruvate, in the cytoplasm, with or without oxygen

• glucose broken in two:

• glycolysis takes one 6-carbon glucose and converts it into two 3-C pyruvates

• it runs in the cytoplasm of every cell that has one — no organelle required

• it works with oxygen present or absent, which is why it predates aerobic respiration in the evolutionary record

• if oxygen is present, pyruvate can be sent to aerobic metabolism

• if oxygen not present, glycolysis keeps going through fermentation

big picture of glycolysis

two phases

• glycolysis spends ATP before it earns ATP

• phase 1: energy investment

  • steps 1 through 5

  • spends 2 ATP

  • the cell pays up front to phosphorylate glucose and prime the molecule for splitting

    • the phosphorylations trap glucose inside the cell and make the six-carbon molecule easier to break

• phase 2: energy payoff

  • steps 6 through 10

  • earns 4 ATP + 2 NADH

  • both 3-C halves are processed in parallel, each yielding 2 ATP and 1 NADH

net gain: 2 ATP per glucose

phase 1

• GAP continues directly into phase 2 but DHAP gets rapidly converted into GAP through triose phosphate isomerase

  
  

• one glucose has become two GAP molecules, each three carbons and the cell is down 2 ATP

phase 2

• starts with GAP and ends with pyruvate

• oxidation of GAP makes NADH

• first ATP made when high-energy phosphate is transferred to ADP, and the second in the final step when PEP donates its phosphate to ADP

• both halves run in parallel. the x2 amplifier

• phase 2 total: 4 ATP + 2 NADH (2 from each 3-C track)

the ledger

• gross yield minus the up-front investment

• net per glucose: 2 ATP +2 NADH. modest, but glycolysis only takes seconds and runs anywhere, even in cells without oxygen

first 5 steps; phase 1

• ATP spent (steps 1 and 3)

• 3 reversible rearrangements

• end with 2 GAPs ready for phase 2

• step 1: irreversible regulatory step: ATP spent when phosphorylating glucose to G-6-P by hexokinase

• step 2: phosphoglucose isomerase: G-6-P → F-6-P

• step 3: irreversible regulatory step: ATP spent by phosphofructokinase-1 (PFK1): F6P → F-1,6-BP

• step 4: aldose: splits the six-carbon molecule into DHAP and GAP

• step 5: triose phosphate isomerase-catalyzed: DHAP → GAP so by the end of the pathway, 2 GAP molecules are produced

step 1

hexokinase: induced fit

• glucose binds first — the enzyme closes around it and positions ATP

• closure positions ATP next to glucose C6 and shuts out water, so ATP is not wasted by hydrolysis

  • the enzyme is trying to transfer the terminal phosphate to glucose rather than hydrolyze ATP wastefully

• produces G-6-P + ADP — the phosphate gives the G-6-P a negative charge, helping to trap it in the cell and mark it for metabolism by that cell

trapped, and always primed

• G6P’s negative charge prevents leaving.

• Km ≈ 0.1 mM vs intracellular glucose 1 to 5 mM means hexokinase is saturated

• once glucose enters the cell, hexokinase is ready to phosphorylate it, marking it for use very quickly

induced fit = safety mechanism. no glucose, no closed site, no wasted ATP

steps 2

isomerization

• phosphoglucose isomerase

• G6P → F6P (aldose to ketose)

• constitutional isomers, same formula, different connectivity.

• no ATP cost, freely reversible

• structural reason: fructose form sets up the molecule for the later cleavage into two 3-C pieces

step 3

committed step

• control point

• phosphofructokinase-1 (PFK-1)

• F6P → F1,6BP w/ ATP

• rate limiting and committed

• once PFK-1 adds the second phosphate, the cell has no use for the molecule except glycolysis

  • G6P still has options → glycolysis, glycogen metabolism, or pentose phosphate pathway

PFK-1 is the most regulated enzyme in glycolysis

step 4

cleaves F1,6BP in half

• aldose — cleaves between C3 and C4 to produce DHAP and GAP

step 5

coverts DHAP to GAP

• triose phosphate isomerase

• result: 2 GAP enter phase 2

  • Le Chatelier: as phase 2 consumes GAP, TPI pulls more DHAP across even through the mixture actually favors DHAP

  • effectively, both halves run

• from this point on, every reaction happens twice per glucose (because 2 GAP produced)

steps 6-10: phase 2

• step 6: GAPDH (glyceraldehyde 3-phosphate dehydrogenase): NAD+ reduced to NADH

• step 7: phosphoglycerate kinase: makes first ATP of phase 2 by substrate level phosphorylation

• step 8: phosphoglycerate mutase: moves the phosphate from C-3 onto C-2

• step 9: enolase: removes water and creates PEP (phosphoenolpyruvate) which contains a very high energy phosphate donor

• step 10: irreversible regulatory step: pyruvate kinase: PEP donated its phosphate to ADP to make the second ATP and pyruvate

step 6

• GAPDH does two things at once: oxidizes the aldehyde and phosphorylates it in one coupled reaction

  • oxidizes the aldehyde group of GAP. the electrons removed from GAP are transferred to NAD+ making NADH

• the chemistry:

  • GAP’s aldehyde is oxidized to a carboxyl-like group

  • Pi from solution is added at the same time, trapping the energy released by the oxidation as an acyl phosphate bond

  • the electrons stripped off GAP go onto NAD+, making NADH

  • at the same time, the dehydrogenase attaches inorganic phosphate from solution to oxidized carbon, making 1,3-bisphosphoglycerate

  • important feature: acyl phosphate at carbon one because acyl phosphates have a high phosphoryl-transfer potential and hydrolyzing this bond releases more free energy than it does hydrolyzing ATP

  why it matters:

  • 1,30BPG is a high-energy intermediate. its phosphate is set up to be donated to ADP in step 7, making ATP

  • without the energy stored here, step 7 couldn’t make ATP at all

  • this substrate-level phosphorylation: ATP made directly from a high-energy substrate, not from the electron transport chain

GAPDH couples oxidation to phosphorylation. it pre loads the energy that step 7 will collect as ATP

step 7

• phosphoglycerate kinase

• PGK transfers the high-energy phosphate from 1,3-BPG onto ADP, making ATP and the 3-phosphoglycerate carbon product

• this is the first step that glycolysis produces

step 8

• phosphoglycerate mutase

• the phosphate moves from carbon 3 to carbon 2 to make 2-phosphoglycerate

• this is a setup move: 2-PG is the right substrate for enolase, the next enzyme

step 9

• enolase removes a water

• the resulting phosphoenolpyruvate (PEP) holds an enol phosphate, one of the highest-energy phosphate bonds

  • PEP= high phosphoryl- transfer potential because after phosphate transfers, the enol form of pyruvate can tautomerize to the more stable keto form

• by the end of this step, the substrate that is going to drive the final ATP producing step is created

step 10

• PEP donates its phosphate. ATP out. Pyruvate locked in

why irreversible:

• PEP releases ~62 kJ/mol when it loses its phosphate. only ~31 kJ/mol is needed to make ATP from ADP

• pyruvate is stabilized by tautomerization

• the extra energy is released as heat, locking the reaction one-way

the third regulatory step:

• like hexokinase and PFK-1, pyruvate kinase is irreversible.

• its regulated allosterically and hormonally, in tissue-specific ways

total irreversible steps:

• hexokinase (step 1)

• PFK-1 (step 3)

• pyruvate kinase (step 10)

these are the regulatory checkpoints

where regulation lives

• the three irreversible steps from module 2 are the regulatory checkpoints

• muscle: senses energy; ATP/AMP at PFK-1. glycolysis runs when energy is low

  • ATP needed for contraction and often needed immediately

  • regulation is built around energy charge, so presence of ATP, ADP and AMP

• liver: senses fuel; glucokinase +F-2, 6-BP. glycolysis runs when glucose is plentiful

  • managed blood glucose for the entire body — after a meal, takes it glucose and processes it but during fasting, it should preserve or release glucose instead of burning it itself

the same three enzymes regulate in both tissues. the signals they listen to are tissue-specific

• muscle mainly listens to its own energy state

• liver listens to glucose availability and whole-body hormonal cues

muscle strategy

muscle regulatory control centers rely on PFK-1 (glycolysis step 3), so PFK-1 is inhibited by ATP — if ATP is abundant, the cell doesn’t need to burn glucose rapidly for more ATP. PFK-1 is also activated by AMP — rises sharply when ATP is consumed faster than it is being regenerated

• opposite signals, exquisite sensitivity

• ATP inhibits PFK-1; AMP relives inhibition and activates glycolysis when energy is low

• the trick: one enzyme, opposite signal

  • PFK-1 integrates ATP and AMP through allosteric regulation

  • high ATP favors a low-activity state; rising AMP shifts the enzyme toward high activity

• why the ratio matters:

  • ATP is abundant at rest, AMP = low

  • AMP is a sensitive low-energy signal. cellular ATP greatly exceeds AMP at rest, so even a small ATP drop can produce a large relative AMP increase and rapidly disinhibit PFK-1

    • through adenylate kinase reaction — makes AMP an amplified signal

energy charge falls → AMP rises → PFK-1 turns on → glycolysis surges

muscle: rest vs exercise

• muscle PFK-1 flips between off and on as ATP/ AMP swings

• this is fast, local, no hormones involved

• at rest, ATP= plentiful and AMP low

• ATP inhibits PFK-1, so step 3 slows

• when downstream flux is slow, G6P starts to accumulate, which will feedback inhibit hexokinase (the enzyme that produces it)

• during exercise, ATP is being used rapidly, so AMP rises and PFK-1 becomes more active

• more F6BP produced and upstream product will also activate pyruvate kinase

• that feed-forward activation helps the end of the glycolysis pathway keep pace with the beginning

hexokinase feedback

• G6P inhibits its own production

• if the cell doesn’t need more G6P, hexokinase backs off

• depends on what happens downstream at PFK-1

• at rest: PFK-1 is slow → G6P piles up → G6P inhibits HK → glucose entry stalls

• during exercise: PFK-1 is fast → G6P stays low → HK is freed → glucose pours in

PFK-1 sets the pace. hexokinase follows along through the G6P feedback loop

• prevents the cell from wasting ATP on glucose phosphorylation when the rest of glycolysis isn’t ready to use the product

liver strategy (VIDEO 3 AT 6:35)

• the liver listens to glucose, not ATP

• its job is to buffer the body’s blood sugar, not to make its own ATP

• muscle (recap) : signal= energy charge

  • ATP inhibits PFK-1; AMP relieves it

  • HK has low Km, saturated at normal glucose

  • glycolysis runs when the cell needs ATP

  • goal: keep ATP up

• liver: signal= glucose abundance

  • different step 1 enzyme: glucokinase (HK IV)

  • high km → activity rises with blood glucose

  • PFK-1 controlled by F-2,6-BP (hormone-linked)

  • citrate inhibits PFK-1 here too (TCA fuel signal)

  • goal: keep ATP up

• liver jobs:

  • manage blood glucose for the entire body

  • after a meal: blood glucose and insulin levels begin to rise so liver should take up glucose, burn some and store some as glycogen and covert excess carbon into fat

  • during fasting, glucagon levels rise and liver should stop burning glucose, break down glycogen, make glucose by gluconeogenesis and release glucose into the blood stream for the rest of the body to use

  • should be able to respond to glucose abundance and hormones more so than local ATP demand — done by glucokinase (step 1) and fructose 2,6 -bisphosphate (regulates PFK-1)

same pathway, different mandate. muscle works for itself; liver works for the body

liver step 1

• uses glucokinase (instead of hexokinase like muscles) (also sometimes called hexokinase IV)

• glucokinase has a different Km (around 8 mM)

• high Km means glucokinase activity rises with blood glucose across the normal range

  • not saturated at normal blood glucose levels so at around 5 mM glucose, glucokinase is active, but still has room to increase

• what this means:

  • at normal blood glucose (~5mM):

    • hexokinase is already saturated. muscle uses glucose freely

    • glucokinase is only at about 40% of Vmax. liver pulls in glucose gradually

  • after a meal (~10mM)

    • glucokinase rises close to Vmax

    • liver glucose uptake accelerates exactly when blood glucose is high. that’s the buffering role

higher Km means liver glucose uptake tracks blood glucose, instead of always running flat-out

liver PFK-1

• different regulators, different signals

• F-2,6-BP is the dominant activator. citrate is the brake

• new in liver compared to muscle: F-2,6-BP, a hormone-controlled metabolite

• citrate, the downstream fuel signal, brakes PFK-1 in both tissues

liver PFK-1 is especially shaped by F-2,6-BP, on top of ATP/ AMP and citrate

three hormones, three states

• Insulin (fed state)

• glucagon (fasted state)

• epinephrine (stress/ emergency)

the body choses on hormonal state at a time. each one routes through PFK-2 and pyruvate kinase in the liver

Insulin

• fed state — after a meal

• “burn the incoming glucose. store the surplus”

• liver glycolysis: ON

• liner glycogen synthase: ON

• muscle glucose uptake: ON (GLUT4)

glucagon

• fasted state — blood glucose dropping

• “make and release glucose. Don’t burn it”

• liver glycolysis: OFF

• liver gluconeogenesis: ON

• muscle: no response (no receptor)

epinephrine

• stress/ emergency — fight, flight, sprint

• “mobilize glucose to muscle, fast”

• liver glycolysis: OFF (save glucose)

• liver glycogenolysis: ON

• muscle glycogenolysis: ON

the F-2,6-BP switch

• one enzyme, two activities. a phosphate flips it

• PFK-2 builds F-2,6-BP from F6P

• FBPase-2 tears it down —F -2,6 -BP → F6P

• same protein, different states

• phosphorylation status of one enzyme determines whether F-2,6-BP is being built or destroyed

• when liver enzyme is dephosphorylated, PFK-2 activity dominates (F-2,6-BP levels rise and PFK-1 is activated)

• when the enzyme is phosphorylated, FBPase-2 activity dominates (F-2,6-BP levels fall and PFK-1 loses its strongest activator)

insulin in the liver

• dephosphorylate everything, burn the glucose

• insulin signals fed state. the liver shifts towards glycolysis

• result: liver glycolysis is fully turned on. two of the three regulatory enzymes (PFK-1 and PK) are activated together

• insulin → dephosphorylation → PFK-1 on + PK on → liver burns the incoming glucose

• turns on step 3 and 10

glucagon in the liver

• phosphorylate everything, save the glucose

• glucagon signals fasted state. the liver shuts glycolysis down to release glucose to the body

• result: liver glycolysis is shut down. the liver now releases glucose (gluconeogenesis and glycogenolysis run instead)

• glucagon → phosphorylation → PFK-1 off + PK off → liver makes and releases glucose, doesn’t burn it

• cAMP levels rising by the GPCR receptor signaling pathways

• shuts down steps 3 and 10

muscle’s different game: same hormone, different receivers

• muscle has no glucagon receptor. insulin and epinephrine do different things

• muscle glycolysis is mostly allosteric. hormones in muscle act on transporters and glycogen, not on PFK-1 directly

• in liver, PKA can inhibit pyruvate kinase by phosphorylation

• skeletal muscle expresses a different pyruvate kinase isozyme that is not regulated in the same way so epinephrine mobilizes fuel in muscle without shutting off the glycolytic endpoint

big idea

• muscle hormones control access to fuel

• local ATP and AMP largely control the glycolytic enzyme activity

insulin response

• GLUT4 to the surface

• insulin doesn’t directly hit PFK-1 or pyruvate kinase in muscle

• instead, it triggers translocation of GLUT4 glucose transporters to the muscle cell membrane

• more glucose enters, allosteric regulation handles the rest

glucagon response

• no response

• muscle cells lack a functional glucagon receptor used for glucose metabolism

• glucagon circulates past the muscle without binding

• glucagon is liver-specific in its effects on glucose metabolism

epinephrine response

• glycogenolysis

• epinephrine binds muscle β-adrenergic receptors and activates PKA, which mobilizes muscle glycogen

• glucose-1-P enters glycolysis after conversion to G6P

• glycolysis floods with fuel; ATP gets made fast

muscle summary

• sense self

• role: make ATP for contraction

• primary signal: energy charge (ATP/AMP) at PFK-1

• hexokinase: low Km, saturated at normal glucose

• insulin moves GLUT4 to membrane

• epinephrine: drives glycogen breakdown

• glucagon: no receptor, no response

liver summary

• sense the body

• role: buffer blood glucose

• primary signal: blood glucose via glucokinase (high Km)

• F-2,2-BP hub: PFK-2 phosphorylation routes hormones in

• insulin: dephosphorylates, glycolysis ON

• glucagon/ epinephrine: phosphorylates, glycolysis OFF
  net result: liver releases glucose when fasted

pyruvate has two roads

• same starting pyruvate, two destinations. oxygen makes a 15-fold difference in ATP yield

why fermentation exists

• the cell runs out of NAD+ first

• GAPDH needs NAD+ at step 6 of glycolysis. without it, the pathway stalls

  • it oxidizes GAP and reduces NAD+ to NADH

• the problem: the NAD+ pool is small, two GAPDH turns per glucose drain it fast. without reoxidation, GAPDH stalls and glycolysis stops

• aerobic solution: shuttle NADH electrons into mitochondria. the ETC oxidizes them and regenerates NAD+

• fermentation solution: uses pyruvates itself as the electron acceptor. reducing it consumes NADH and regenerates NAD+

lactate fermentation

• one step, one enzyme

• lactate dehydrogenase reduces pyruvate to lactate, regenerating NAD+

• per glucose, 2 pyruvates are reduced to 2 lactates and 2 NADH are converted back to 2 NAD+ so GADPH keeps running

sprinting muscle

• temporary anaerobia

• during intense effort, ATP demand outruns oxygen supply

• muscle fibers shift to lactate fermentation, producing ATP fast while lactate builds up

• once oxygen returns, the lactate is shuttled to the live via the bloodstream (Cor cycle)

red blood cells

• permanent anaerobia

• RBCs lack mitochondria entirely (no room and they shouldn’t consume the oxygen they are delivering)

• glycolysis + lactate fermentation is their only ATP source, 24/7

• the lactate is released to the blood for the liver to recycle

one enzyme. one step. the pyruvate becomes the dump site for the NADH electrons

ethanol fermentation

• two steps, yeast’s solution

• decarboxylate first, then reduce. same NAD+ regeneration, different product

the chemistry:

• step one strips off CO2 as a gas. this is irreversible, uses no NAD+ and is not a redox step

  • uses thiamine pyrophosphate as a cofactor

• step two is the actual reduction: NADH delivers two electrons to acetaldehyde, producing ethanol and regenerating NAD+

where it happens:

• yeast and some bacteria — brewing, baking and wine

• the CO2 is what makes bread rise and beer foam

• the ethanol is the byproduct humans founds use for

• animals do not have pyruvate decarboxylase; we cannot make ethanol from glucose so we reduce pyruvate to lactate instead /

different products, same NAD+ logic. the reduction step in both fermentation is the point

the price of no oxygen

• fermentation is a 15-fold pay cut

• same glucose, same glycolysis. the difference is what happens after

• the ETC produces the vast majority of ATP. without oxygen, the ETC can’t run and you’re left with just substrate-level

fermentation gives up the TCA cycle and oxidative phosphorylation: about 2 vs ~30 ATP per glucose

• fermentation trades efficiency for speed and independence from oxygen — useful for sprinting muscle and essential for red blood cells

where the lactate goes

• the Cori cycle: muscle and liver split the work

  • muscle converts glucose to lactate and exports it to the blood stream

  • liver takes up lactate and oxidizes it back to pyruvate and then liver uses gluconeogenesis to rebuild glucose which can be released back into the bloodstream

• lactate isn’t waste. it’s a shuttle.

• net cost: muscle gets fast 2 ATP without waiting for oxygen. liver pays the metabolic bill with its own 6 ATP later (to remake one glucose from 2 lactate)

• lactate is a metabolic loan. muscle borrows speed; liver repays in oxygen and ATP later

fructose in liver

• the bypass route

• hepatic fructose enters glycolysis below hexokinase and PFK-1

clinical implication:

• bypassing PFK-1 means no allosteric brake on hepatic fructose metabolism

• fructose flux runs largely unregulated. excess fructose floods the liver with triose phosphates, which feed acetyl-CoA and de novo lipogenesis

• high chronic intake is a major driver of non-alcoholic fatty liver disease (NAFLD)

liver fructose enters glycolysis past the gatekeepers. excess fructose becomes fat

• bad when high fructose concentrations

fructose in muscle and adipose

• outside the liver, fructose is phosphorylated by hexokinase to F6P and enters glycolysis normally

why the difference from the liver:

• muscle and adipose do not express fructokinase

• their only entry point for fructose is hexokinase, which makes F6P. that F6P then must pass through PFK-1, which is fully regulated by ATP, AMP, and citrate

• hexokinase has low affinity for fructose, so the rate is limited. combines with the PFK-1 gate, extrahepatic fructose metabolism is slow and well-controlled

outside the liver, fructose plays by glucose’s rules. PFK-1 still decides how fast it gets used

galactose: the Leloir pathway

• three steps, four enzymes, one disease

• dietary galactose (from milk lactose) is converted to G6P and enters glycolysis at step 2

clinical: galactosemia

• most common form: classic galactosemia (type I), a GALT deficiency

• without GALT, Gal-1-P accumulates and it toxic to liver, kidney and brain

• infants fed milk develop jaundice, hepatomegaly and intellectual disability if untreated

• treatment: life-long galactose-restricted diet

• newborn screening catches it before symptoms appear

galactose joins glycolysis as G6P. GALT deficiency makes it medically dangerous