Glycolysis

Explain the biological purpose of glycolysis in organisms that have access to oxygen versus those that do not

Provides ATP quickly; aerobic cells funnel pyruvate → TCA/ETC, anaerobic cells use fermentation to regenerate NAD⁺

Explain why glycolysis is considered both an energy-producing and a biosynthetically important pathway

Produces ATP/NADH and supplies intermediates for amino acids, lipids, nucleotides

Explain why glycolysis is evolutionarily conserved across nearly all forms of life

Does not require O₂ or organelles; ancient, efficient ATP generation

Explain why glycolysis must be tightly regulated despite being energetically favorable overall

Prevents wasteful ATP consumption and futile cycling; matches energy demand

Explain why glycolysis can proceed under anaerobic conditions but oxidative phosphorylation cannot

Glycolysis uses substrate-level phosphorylation; OxPhos requires O₂ as terminal electron acceptor

Explain why cells require NAD⁺ for glycolysis to proceed

NAD⁺ accepts electrons in GAPDH step; required for continued carbon oxidation

Explain why NADH must be reoxidized to NAD⁺ for glycolysis to continue

NAD⁺ pool is limited; must be regenerated via ETC or fermentation

Explain why glycolysis uses NAD⁺ rather than FAD as an electron carrier

NAD⁺ accepts high-energy electrons; FAD used for lower-energy redox reactions

Explain the biochemical consequences if NAD⁺ regeneration is blocked

GAPDH halts → glycolysis stops → ATP depletion

Explain how the NADH/NAD⁺ ratio influences metabolic flux through glycolysis

High NADH inhibits GAPDH; high NAD⁺ promotes glycolytic flux

Explain the catalytic strategy used by triose phosphate isomerase (TIM)

General acid–base catalysis via Glu/His forming enediol intermediate

Draw and explain the enediol intermediate formed during the TIM-catalyzed reaction

Proton abstraction forms enediol; reprotonation yields isomerized triose

Explain why TIM is considered a “catalytically perfect enzyme”

Rate limited only by substrate diffusion

Describe the structure of TIM and explain how its structure contributes to catalysis

TIM barrel positions catalytic residues optimally and excludes water

Explain the role of general acid–base catalysis in glycolysis, using a specific enzyme as an example

TIM uses Glu as base and His as acid to shuttle protons

Explain how induced fit is used by enzymes in glycolysis to stabilize transition states

Substrate binding causes conformational change that lowers activation energy

Explain the role of His, Glu, Asp, and Lys residues in glycolytic enzyme active sites

His/Glu/Asp act in proton transfer; Lys stabilizes negative charges

Explain what a Schiff base is and how it is formed in enzyme catalysis

Covalent imine between Lys ε-NH₂ and carbonyl substrate

Draw a Schiff base between an enzyme and a glycolytic intermediate and label all functional groups

Imine linkage between Lys and carbonyl carbon stabilizes carbanion

Explain why Schiff base formation stabilizes carbanion intermediates

Delocalizes negative charge through covalent enzyme linkage

Explain which enzyme(s) in carbohydrate metabolism use Schiff base catalysis and why

Aldolase; stabilizes carbanion during C–C bond cleavage

Explain how Schiff bases lower activation energy during aldose–ketose interconversion

Stabilize high-energy intermediates and align reactive groups

Explain the difference between keto–enol and enediol intermediates

Enediol has two hydroxyls and double bond; keto–enol involves proton shift

Explain how tautomerization is used as a catalytic strategy in glycolysis

Enolization allows rearrangement of carbonyl position

Explain why isomerization reactions are necessary in glycolysis

Allows symmetric cleavage and energy extraction

Explain how proton abstraction and donation are coordinated during isomerization reactions

Active-site acids/bases shuttle protons in defined sequence

Explain why the hydrolysis of phosphoenolpyruvate (PEP) is more favorable than ATP hydrolysis

Enol → keto tautomerization of pyruvate drives reaction

Explain how tautomerization of pyruvate contributes to the large negative ΔG

Product stabilization pulls equilibrium forward

Explain why certain glycolytic steps are essentially irreversible in cells

Large negative ΔG; far from equilibrium

Explain how ATP investment and payoff phases are balanced in glycolysis

Early ATP consumption enables later high-yield ATP production

Explain how coupling unfavorable reactions to favorable ones allows glycolysis to proceed

ATP hydrolysis drives endergonic steps

Explain why phosphofructokinase-1 (PFK-1) is the key regulatory enzyme of glycolysis

First committed, irreversible step

Explain how ATP both serves as a substrate and an allosteric inhibitor of PFK-1

High ATP binds inhibitory site, lowering affinity for F6P

Explain how AMP and ADP regulate glycolysis and what they signal about cellular energy status

Activate PFK-1; signal low energy

Explain the role of citrate in regulating glycolysis

Inhibits PFK-1; signals abundant biosynthetic precursors

Explain how fructose-2,6-bisphosphate regulates glycolysis

Potent PFK-1 activator; overrides ATP inhibition

Explain the reaction catalyzed by PFK-2

F6P + ATP → F2,6BP + ADP

Explain how PFK-2 differs from PFK-1 in function and regulation

PFK-2 makes regulator, not glycolytic intermediate

Explain how hormonal signaling alters PFK-2 activity

PKA phosphorylation shifts activity toward FBPase-2

Explain how phosphorylation affects PFK-2/FBPase-2 activity

Phosphorylation inhibits PFK-2, activates FBPase-2 (liver)

Explain how glucagon signaling alters glycolytic flux in liver cells

↓ F2,6BP → ↓ glycolysis, ↑ gluconeogenesis

Explain how insulin signaling alters glycolytic flux

↑ F2,6BP → ↑ glycolysis

Explain why cancer cells with mutated PFK-2 exhibit increased glycolysis

Constitutively high F2,6BP activates PFK-1

Explain why glycolysis and gluconeogenesis cannot operate simultaneously at high rates

Futile cycle wastes ATP

Explain the concept of reciprocal regulation between glycolysis and gluconeogenesis

Same signals activate one pathway and inhibit the other

Explain why bypass reactions are required for gluconeogenesis

Irreversible glycolytic steps must be circumvented

Explain how fructose-2,6-bisphosphate coordinates glycolysis and gluconeogenesis

Activates PFK-1, inhibits FBPase-1

Explain the purpose of fermentation in cells

Regenerates NAD⁺ without O₂

Explain how lactic acid fermentation regenerates NAD⁺

Pyruvate reduced to lactate using NADH

Explain how alcohol fermentation regenerates NAD⁺

Acetaldehyde reduced to ethanol using NADH

Explain why fermentation does not yield additional ATP beyond glycolysis

No electron transport or proton gradient

Explain why fermentation is essential for organisms lacking mitochondria

Only method to regenerate NAD⁺

Explain why arsenate is a poison and how it interferes with glycolysis

Replaces Pi in GAPDH step → unstable product → no ATP formed

Explain how arsenate affects ATP yield without stopping glycolysis entirely

Glycolysis continues but substrate-level phosphorylation bypassed

Explain how silver poisoning interferes with enzyme function

Binds thiol groups → enzyme inactivation

Explain how superoxide formation can inhibit the pyruvate dehydrogenase complex

Damages Fe-S clusters and lipoamide

Predict the metabolic consequences of inhibiting GAPDH

ATP depletion; upstream metabolite buildup

Explain what would happen to cellular metabolism if GAPDH could not use NAD⁺

Glycolysis stops; cells rely on alternative ATP sources

Explain which steps of glycolysis are most sensitive to oxidative stress and why

GAPDH, PDH; redox-sensitive cofactors

Draw the active site of a glycolytic enzyme and label key catalytic residues

Includes His/Glu/Asp for acid-base catalysis, Lys for charge stabilization

Identify nucleophilic residues involved in glycolytic enzyme mechanisms

Ser, Cys, Lys

Explain how functional group chemistry enables carbon–carbon bond rearrangements

Carbanion stabilization via Schiff bases/enediols

Explain how enzymes stabilize negatively charged intermediates during glycolysis

Electrostatic interactions and metal ions

Trace the fate of all six carbons of glucose through glycolysis

Split into two identical 3-carbon pyruvates

Explain why carbon rearrangement is necessary before cleavage in glycolysis

Ensures equal energy extraction

Explain how symmetry influences carbon fate after triose formation

Carbons become equivalent after DHAP ↔ GAP

Explain how glycolysis connects to the TCA cycle

Pyruvate → acetyl-CoA → TCA

Explain how glycolysis connects to the pentose phosphate pathway

G6P diverted to PPP

Explain the role of 2,3-bisphosphoglycerate and why it is not an intermediate of glycolysis

Regulates hemoglobin O₂ binding; branch off pathway

Explain why glycolytic intermediates are useful precursors for biosynthesis

Provide carbon skeletons

Explain everything you know about the regulation of glycolysis in liver cells

Allosteric control (ATP, AMP, citrate), hormonal control via PFK-2/F2,6BP

Explain everything you know about glycolysis in the context of whole-body metabolism

Fed state energy supply; fasting shifts to gluconeogenesis

Explain how glycolysis adapts to fed versus fasted states

Fed: insulin ↑ glycolysis; fasted: glucagon ↓ glycolysis

Explain how enzyme structure, catalysis, regulation, and energetics are integrated in glycolysis

Structure enables catalysis; regulation matches energy demand

Explain why glycolysis is indirectly dependent on oxidative phosphorylation

Requires NAD⁺ regeneration

Explain how uncoupling oxidative phosphorylation affects glycolysis

↑ NAD⁺ regeneration → ↑ glycolysis

Explain how ATP demand influences glycolytic rate

High ADP/AMP activate PFK-