MCB 2000 Chapter 15

cells need energy for:

• mechanical: cilia beating, flagella moving, organelles traveling around the cytoskeleton, chromosomes separating during mitosis

• active transport: Na/K/ATPase

• biosynthesis: proteins from amino acids, nucleic acids from nucleotides, lipids from smaller precursors, polysaccharides from sugars

metabolism is a network

• pathways feed each other

• almost nothing happens in isolation

• central molecules like acetyl-CoA serves as metabolic crossroads

• carbs, fats and proteins all enter as fuel— their carbon skeletons funnel towards central hubs (especially acetyl-CoA) and from there, carbon enters the TCA cycle and feed energy production through the electron transport chain

• same intermediates can also be used to make new intermediates — TCA intermediates, pyruvates, and acetyl-CoA are not just fuel molecules, they’re crossroads

• catabolic and anabolic pathways share intermediates and even enzymes and compartments

catabolism

• breaking things down, releasing energy

• ex. glucose → Co2 + H2O

• one big ordered molecule (six carbons) becomes six tiny CO2 molecules and water

• thermodynamically a slam dunk, ΔG is strongly negative

• the cell captures some of the released energy by phosphorylating ADP to ATP and by reducing NAD+ to NADH.

• catabolism is oxidative: carbons lose electrons as C-H bonds become C-O bonds

ATP made

anabolism

• building things up, uses energy

• ex. Amino acids → proteins

• twenty different small molecules get joined into a long, specifically ordered chain with a defined 3D fold

• thermodynamically uphill, ΔG is positive

• the cell drives the reaction by spending ATP and GTP at each step

  • and reducing power (in the form of NADPH)

• anabolism is reductive: carbons typically gain electrons during biosynthesis

ATP used

____ powers _____

catabolism powers anabolism

both run continuously

amphibolic

• a pathway that serves as both catabolism and anabolism. It oxidizes substrate to release energy and supplies intermediates for biosynthesis

• the TCA cycle is the canonical example

  • acetyl-CoA enters, carbon is oxidized to CO2 and cycle generates NADH and FADH2 that help make ATP

  • TCA intermediates also leave the cycle to build other molecules — make amino acids, heme (the iron-containing group in hemoglobin and cytochromes), citrate helps supply acetyl-CoA for fatty acid and cholesterol synthesis

• the TCA cycle is not just a furnace, it is also a parts depot

three pathway topologies

• linear

• cyclic

• branched

linear pathway

• linear converts

• input in, output out

• once a molecule passes through, the pathway doesn’t see it again

cyclic pathway

• cycles regenerate

• one oxaloacetate processes thousands of acetyl-CoA, components are catalysts, not consumed

• in the TCA cycle, oxaloacetate is regenerated each turn of the cycle, so a small pool of intermediates can process a steady stream of acetyl-CoA

branched pathway

• branches allocate

• one precursor fans out to multiple end products

• branch points are decision points — cell can shift flow depending on what it needs

why so many small steps in glucose oxidation

• capture energy in pieces, not all at one

• combustion: all energy released as heat, none captured

• biological oxidation: many capture points; only a few control points

• same total ΔG. cells capture; fires dissipate

• overall reaction releases a huge amount of energy (-2870 kJ/mol)

• heat released is useful, but when released all at once like that, it can’t be used for as much, so smaller steps are used (glycolysis, pyruvate oxidation, TCA cycle and electron transport chain)

• at key points, enzymes couple their smaller drops to ATP, NADH or FADH2 production

compartmentalization

• opposing pathways, kept under separate control

why separate:

• breakdown and synthesis are opposite jobs — burning fat versus building it

• the cell runs them in different places, with different enzymes, and different cofactors

• β- oxidation in the matrix uses NAD+ and FAD to harvest electrons.

• synthesis in the cytosol uses NADPH to supply reducing power

  • so synthesis is not just β- oxidation in the reverse — the cell separates the spaces, enzymes and cofactors

  • separating opposing pathways keeps the cell from building and burning at one

• location is part of regulation

opposite jobs, separate spaces, different cofactors — location is part of regulation

channeling: handing off intermediates

• location control but on a smaller scale — instead of releasing an intermediate into a solution and waiting for it to find the next enzyme, an enzyme complex passes it directly from one active site to the next which improves efficiency, keeps concentrations high locally and protects unstable or reactive intermediates from side reactions

• the pyruvate dehydrogenase complex as the classic example

why channel:

• three active sites bolted into one complex: E2’s lipoyl arm , a long flexible cofactor, swings between them it picks up the acetyl group at E1, hands it to CoA at E2, then visits E3 to be reoxidized

  • E1, 2, 3 are physically assembled into one large complex

• local substrate concentration stays high

• side reactions can’t compete because the intermediate is never in bulk solvent

• unstable intermediates are protected in transit

architecture as regulation, made literal

ATP

• adenosine plus three phosphates in series

• ATP strores and transfers energy

• hydrolysis of the terminal gamma (γ) phosphate drives cellular work

• phosphoanhydride bonds = the high energy bonds

• when ATP is hydrolyzed, water attacks the gamma phosphate and the terminal phosphoanhydride bond breaks, releasing inorganic phosphate (Pi) and leaving ADP

• the bond itself does not contain energy

  • hydrolysis is favorable because the products are more stable — less charge repulsion, more resonance stability

the goldilocks energy level

• ATP sits in the middle of phosphate-transfer ladder

why the middle:

• ATP accepts phosphate from compounds above it on the ladder

• that’s how glycolysis charges ADP back to ATP, PEP and 1,3-BPG are uphill of ATP, so they donate

• ATP donates phosphate to compounds below it

• that’s how biosynthesis spends ATP, glucose-6-P is downhill of ATP, so ATP can phosphorylate glucose

• if ATP was at the top, the cell could not easily regenerate it, but at the bottom would not be able to drive much chemistry

• middle position = universal go-between

charged from above, spent below. ATP is the middle rung

kinetically stable, thermodynamically unstable

• why ATP can sit in solution and still release energy on demand

• ATP sits in your cells in water at about 5mM concentration, ATP hydrolysis is favorable — so why does it not hydrolyze on its own?

the 2 timescales:

• kinetically stability: uncatalyzed hydrolysis of ATP in water is essentially zero. the activation barrier is too high to climb thermally. a solution of ATP sits for hours

• thermodynamic instability: the product sits far below the reactants. when an enzyme provides a path over the barrier, that drop is released as usable energy

ATP holds its energy until an enzyme calls for it

• an enzyme like hexokinase lowers the activation barrier and positions ATP for phosphoryl transfer so the favorable chemistry can have it on command

thermodynamics says where. kinetics says how fast

coupling: how ATP drives unfavorable reactions

• Add the ΔG°' values. the combined reaction must be negative

• add the standard free energies. if the sum is negative, the couples reactions run

peptide bond formation

when you need to flip a half-reaction

• as given in the text: alanylglycine + H2O → alanine + glycine; ΔG°' = -29 kJ/mol

• real cells spend several high-energy bonds per peptide. just barely is not enough

ATP : universal and rapid

universal:

• every domain of life uses ATP

• bacteria, archaea, fungi, plants, animals, every cell on earth uses ATP as its primary energy currency

• three and a half billion years of evolution and nothing has replaced it. the same molecule powers an E.coli dividing in a flask and a neuron firing in your visual cortex right now

currency, not capital:

• spent and re-earned, second by second

• cells don’t store ATP, they regenerate it constantly.. humans cycle roughly their own body weight in ATP every 24 hours

• the standing pool of ATP would last only seconds under heavy demand. the cell holds its energy as flux, not as a stockpile

three and a half billion years and nothing has replaced ATP

fuel= reduced carbon

• carbon with lots of C-H bonds has lots of electrons to give up

the principle:

• fuels line near the bottom of the ladder. carbs sit in the middle. fatty acid tails sit at the very bottom, long hydrocarbon chains packed with C-H bonds (fatty acids — long chains of CH2 with very little oxygen)

• reduced carbon is energy rich because it has electrons to give up

• catabolism oxidizes that carbon towards CO2

• oxidation releases energy: catabolism walks the carbon upward, transferring electrons to NAD+ or FAD at each step

• more rungs to climb= more ATP per gram. that’s why fats yield more than carbs

reducing carbon has electrons to give. catabolism walks carbon up to CO2

three fuels, one engine

• different inputs, common downstream pathways

• three inputs, three preparations pathways, one common engine. Almost all of the ATP comes from the last two stages, not from the prep work

• most roads lead to acetyl- CoA. all lead to the TCA cycle, then the ETC

• carbs: some electrons captured as NADH

• fats: some electrons captured as NADH and FADH2

• shared engine is not just about carbon flow, but about electron flow

  • acetyl-CoA enters the TCA cycle and continues to oxidize carbon into CO2, and the released electrons are loaded onto NADH and FADH2

  • those carriers deliver the electrons to the ETC where oxygen is the final acceptor and ATP synthase captures the energy as ATP

three stages of catabolism

• digestion/ hydrolysis

• conversion to acetyl-CoA

• TCA + ETC + ATP synthase

digestion/ hydrolysis

• ≈ 0 ATP

• big polymers → monomers

• starch → glucose

• triglycerides → fatty acids+ glycerol

• proteins → amino acids

• happens in the gut, lysosomes, or extracellular space

conversion to acetyl- CoA

• small ATP yield

• glycolysis (glucose → pyruvate → acetyl-CoA )

• β-oxidation (fatty acids → acetyl-CoA)

• deamination (amino acids → various)

• some ATP and NADH already generated here

TCA + ETC + ATP synthase

• ~90% of total ATP

• acetyl- CoA enters the TCA cycle

• NADH and FADH2 donate electrons to the ETC, which pumps protons and drives ATP synthase

• this is where most of the ATP is made

most of the ATP comes from stage 3. the earlier stage mainly prepares the fuel

activated carriers do the work

• ATP: phosphoryl groups — adenosine + 3 phosphates. donates phosphate to substrates, driving uphill reactions

• NADH/ FADH2: electrons (high energy) — reduced cofactors. donate electrons to the ETC, which is where most ATP gets made

• Acetyl- CoA: two carbon acyl units — carriers activated acetyl into the TCA cycle. the hub of carbon catabolism

other cofactors:

• NADPH: biosynthetic electrons (anabolism)

• CoA: acyl-group carrier (fatty acid metabolism)

• TPP: decarboxylation

• biotin: carboxyl-group carrier

same handful of carriers, every pathway. learn them once, recognize them everywhere

recurring reaction types

most of catabolism is four kinds of chemistry, repeated

• phosphoryl transfer

• oxidation-reduction

• decarboxylation

• group transfer / isomerization

recognize the chemistry, and every new pathway gets easier

phosphoryl transfer

• ATP + glucose → ADP + glucose-6-P

• ATP donates phosphate to a substrate (or vice versa)

oxidation-reduction

• NAD⁺ + R-CH(OH)-R’ ⇌ NADH + H⁺ + R-CO-R’

• electrons (often as hydrides: H⁻), move between substrate and NAD+/ FAD

decarboxylation

• R-COO⁻ + H⁺ → R-H + CO2

• a carbon leaves as CO2

• common pyruvate, isocitrate, α-KG

• key steps in the TCA cycle

group transfer/ isomerization

• G6P ⇌ F6P or R–OH + R'–COOH → R'–CO–O–R + H₂O

• rearranging a substrate or moving a group from one molecule to another

three mechanisms

• allosteric (seconds)

• covalent (minutes)

• transcriptional (hours)

three timescales. the same enzyme is often regulated by all three at once

• these layers stack

• one enzyme can receive allosteric feedback, medium-term hormonal control and long term transcriptional adjustment all at once

allosteric

• how: a small molecule effector binds away from the active site

• ex. PFK-1: ATP inhibits it, AMP activates it; glycolysis tracks the cell’s ATP level

  • high ATP slows glycolysis while AMP activates it

second: fast, no covalent change

covalent

• how: kinase adds a phosphate; a phosphatase removes it

  • that phosphate acts as a molecular switch

  • major way that hormones like insulin and glucagon coordinate metabolism across tissues

• ex. glycogen synthase phosphorylated is inactive (fasting), dephosphorylated activated (fed)

minutes: hormone-responsive; requires kinase/ phosphatase

transcriptional

• how: change how much enzyme exists: more mRNA, more protein

• ex. insulin induces lipogenic enzymes when fed; glucagon induces gluconeogenic enzymes when fasting

where regulation happens: irreversible steps

• pathways are regulated at their irreversible steps

why here:

• reversible steps run close to equilibrium, so slowing one does nothing

• irreversible steps sit far from equilibrium (large negative ΔG), so changing their rate changes flux.

• a pathway has several and each is a control point

• glycolysis has three: hexokinase, PFK1, and pyruvate kinase

the committed step

• one irreversible step is special: the first one unique to the pathway

• what makes a step “committed”?

  • the first irreversible step unique to a pathway, the point of no return

  • test it: 1) is the step irreversible, and 2) once that step happens, does the molecule only have one fate afterward

many irreversible steps to regulate, but only one committed step per pathway (usually the most heavily regulated one)

• ex. glycolysis: hexokinase is irreversible, but not the committed step because its product (glucose 6-phosphate) can still leave glycolysis (to make glycogen or into pentose phosphate pathway

• PFK-1 is committed because once fructose 1,6-bisphosphate is made, the only road left is to finish glycolysis

three regulatory strategies

different sensors, all at acting one-way regulated steps

• feedback inhibition

• feed-forward activation

• energy charge

three strategies. all act at one-way regulated steps. often layered across a pathway

feedback inhibition

• end product slows the pathway

• logic: the end product inhibits the pathway’s committed step, so flux falls as product builds up

• ex. CTP inhibits ATCase, the committed step of pyrimidine synthesis

  • cell stops making CTP when it already has enough

feed-forward activation

• upstream surge primes the downstream

• logic: an upstream metabolite switches on a downstream enzyme before its substrate arrives

• ex. in glycolysis, F1,6BP activates pyruvate kinase downstream

energy charge

• sense ATP/ ADP/ AMP ratios

• logic: ATP inhibits and AMP activates catabolic enzymes, so energy state sets flux

• ex. PFK-1: ATP inhibits, AMP activates

how the layers integrate

• three layers of control converging on glycolytic flux

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