Metabolism: Glycolysis,

TCA Cycle, and the

ETC-Oxidative Phosphorylation

Metabolism

The sum of the chemical changes that convert

nutrients into energy and the chemically

complex products of cells

Hundreds of enzyme reactions organized into

discrete pathways

Substrates are transformed to products via many

specific intermediates

Metabolic maps portray the reactions

A Common Set of Pathways

Organisms show a marked similarity in their

major metabolic pathways

Evidence that all life descended from a common

ancestral form

There is also significant diversity

Autotrophs use CO2; Heterotrophs use organic

carbon; Phototrophs use light; Chemotrophs use

Glc, inorganics use S and obtain chem energy

through food generated by phototrophs.

The Sun is Energy for Life

Phototrophs use light to drive synthesis of

organic molecules

Heterotrophs use these as building blocks

CO2, O2, and H2O are recycled

Metabolism

Metabolism consists of catabolism and

anabolism

Catabolism: degradative pathways Usually energy-yielding!

“destructive metabolism”

FUELS -> -> CO2 + H2O + useful energy

Anabolism: biosynthetic pathways energy-requiring!

“constructive metabolism” Useful energy + small molecules --> complex molecules

Organization in Pathways

Pathways consist of sequential steps

The enzymes may be:

Separate

Form a multienzyme complex

A membrane-bound system

New research indicates that multienzyme

complexes are more common than once

thought

Catabolism and Anabolism

Catabolic pathways converge to a few end

products

Anabolic pathways diverge to synthesize many

biomolecules

Some pathways serve both in catabolism and

anabolism and are called amphibolic pathways

Digestion of food polymers: enzyme-catalyzed hydrolysis

Glycolysis: glucose catabolism generate ATP without consuming oxygen (anaerobic)

Citric Acid Cycle: metabolism of acetyl-CoA derived from pyruvate, fatty acids, and amino acids acetyl oxidized to CO2 operates under aerobic conditions reduction of coenzymes NAD+ and FAD; energy used to produce ATP

Oxidative phosphorylation: reduction of molecular oxygen by NADH and FADH2 energy of reduced compounds used to pump protons across a cell membrane potential energy of electrochemical gradient drives phosphorylation of ADP to ATP

Comparing Pathways

Anabolic & catabolic pathways involving

the same product are not the same

Some steps may be common to both

Others must be different to ensure that

each pathway is spontaneous

This also allows regulation mechanisms to

turn one pathway and the other off

METABOLIC REGULATION

Regulated by controlling:

1. Amounts of enzymes

2. Catalytic activities

3. Accessibility of substrates

The ATP Cycle

ATP is the energy currency of cells

In phototrophs, light energy is transformed into

the chemical energy of ATP

In heterotrophs, catabolism produces ATP,

which drives activities of cells

ATP cycle carries energy from photosynthesis or

catabolism to the energy-requiring processes of

cells

WHY ATP?

Free energy is released when ATP is

hydrolyzed.

This energy drives reactions that need it

(eg. muscle contraction)

Recall coupled reactions

ATP has a higher phosphoryl transfer

potential

Redox in Metabolism

NAD+ collects electrons released in catabolism

Catabolism is oxidative - substrates lose

electrons, usually H- ions

Anabolism is reductive - NADPH provides the

electrons for anabolic processes, and the

substrates gain electrons

LEO - GER

RECURRING MOTIFS IN METAB

Certain compounds keep on recurring or

appearing in metabolic reactions and their

functions are the same in the processes

Metab looks complicated but reactions are

actually limited and repeating.

ACTIVATED CARRIERS

These species help carry out the

metabolic reactions, even nonfavorable

ones, at times

Example: ATP (activated carrier of

phosphoryl groups)

Activated carriers of electrons for fuel

oxidation: e- acceptors!

Aerobic systems: O2 is the final e-

acceptor, but this does not occur

directly

Fuels first transfer e- to carriers:

pyridine molecules or flavins.

NAD+:

nicotinamide

adenine

dinucleotide

Activated carriers of electrons for fuel

oxidation: e- acceptors!

FAD: Flavin

adenine

dinucleotide

Activated carrier of electrons for

reductive biosynthesis: e- donors!

NADPH: common

electron donor

R is phosphate

group

Activated carrier of two-carbon

fragments

COENZYME A: carrier of acyl groups

Activated carrier of two-carbon

fragments

VITAMINS

Many vitamins are "coenzymes" -

molecules that bring unusual chemistry to

the enzyme active site

Vitamins and coenzymes are classified as

"water-soluble" and "fat-soluble"

The water-soluble coenzymes exhibit the

most interesting chemistry

Key Reactions in Metabolism

1. REDOX reactions

Electron carriers are needed!

2. LIGATION reactions

Bond formation facilitated by ATP cleavage

3. ISOMERIZATION reactions

4.GROUP TRANSFER

5.HYDROLYTIC reactions

Bond cleavage by addition of H2O

6.ADDITION of functional groups to

double bonds or REMOVAL of

groups to form double bonds

Uses lyases

GLYCOLYSIS

Glycolysis

1897: Hans and Eduard Buchner (Sucrose

cell-free experiments; fermentation can

take place outside of living cells)

METABOLISM became simple chemistry

Glycolysis: “Embden-Meyerhof pathway”

The all-important Glucose

The only fuel the brain uses in non-

starvation conditions

The only fuel red blood cells can use

WHY? Evolutionary: probably available for primitive

systems

The products and their fates

AKA Embden-Meyerhof-Parnas Pathway

Involves the oxidation of glucose

Products:

2 Pyruvate

2 ATP

2 NADH

Cytosolic

Glycolysis

Glycolysis

Anaerobic

The entire

process does not

require O2

Glycolysis: General Functions

Provide energy in the form of ATP

Generate intermediates for other pathways: Hexose monophosphate pathway

Glycogen synthesis

Pyruvate dehydrogenase Fatty acid synthesis

Krebs’ Cycle

Glycerol-phosphate (TG synthesis)

Specific functions of glycolysis

Red blood cells (RBCs) Rely exclusively for energy

Skeletal muscle Source of energy during exercise, particularly high

intensity exercise

Adipose tissue Source of glycerol-P for TG synthesis

Source of acetyl-CoA for FA synthesis

Liver Source of acetyl-CoA for FA synthesis

Source of glycerol-P for TG synthesis

Regulation of Cellular Glucose Uptake

Brain & RBC: The GLUT-1 transporter has high affinity for glucose

and is always saturated. Ensures that brain and RBC always have glucose.

Liver: The GLUT-2 glucose transporter has low affinity and

high capacity. Uses glucose when fed at rate proportional to glucose

concentration

Muscle & Adipose: The GLUT-4 transporter is sensitive to insulin

Glucose Utilization

Phosphorylation of glucose

Commits glucose for use by that cell

Energy consuming

Hexokinase: muscle and other tissues

Glucokinase: liver

Regulation of Cellular Glucose

Utilization in the Liver Feeding

Blood glucose concentration high

GLUT-2 taking up glucose

Glucokinase induced by insulin

High cell glucose allows GK to phosphorylate glucose for use by liver

Post-absorptive state Blood & cell glucose low

GLUT-2 not taking up glucose

Glucokinase not phophorylating glucose

Liver not utilizing glucose during post-absorptive state

Regulation of Cellular Glucose

Utilization in the Liver

Starvation

Blood & cell glucose concentration low

GLUT-2 not taking up glucose

GK synthesis repressed

Glucose not used by liver during starvation

Regulation of Cellular Glucose

Utilization in the Muscle

Feeding and at rest High blood glucose, high insulin

GLUT-4 taking up glucose

HK phosphorylating glucose

If glycogen stores are filled, high G6P inhibits HK, decreasing glucose utilization

Starving and at rest Low blood glucose, low insulin

GLUT-4 activity low

HK constitutive

If glycogen stores are filled, high G6P inhibits HK, decreasing glucose utilization

Regulation of Cellular Glucose

Utilization in the Muscle

Exercising Muscle (fed or starved)

Low G6P (being used in glycolysis)

No inhibition of HK

High glycolysis from glycogen or blood

glucose

Regulation of Glycolysis

Regulation of 3 irreversible steps

PFK-1 is rate limiting enzyme and

primary site of regulation.

Regulation of Glycolysis

Most important regulation hub!

Regulation of PFK-1 in Muscle

Allosterically stimulated by AMP High glycolysis during exercise

Allosterically inhibited by ATP

High energy, resting or low exercise

Citrate Build up from Krebs’ cycle

May be from high FA beta-oxidation -> hi acetyl-CoA

Energy needs low and met by fat oxidation

Regulation of PFK-1 in Liver

Inducible enzyme

Induced in feeding by insulin

Repressed in starvation by glucagon

Allosteric regulation

Like muscle w/ AMP, ATP, Citrate

Activated by Fructose-2,6-bisphosphate

Fermentation

Anaerobic respiration!

Produces ATP without oxygen.

No ETC is present since there is no oxygen

NAD+ gets recycled by use of an organic hydrogen acceptor like lactate or ethanol.

Common in prokaryotes and very useful to humans.

Fermentation

Two type lactic acid and alcohol fermentation.

A build up of lactate in your muscles from over exerting yourself and not taking in enough oxygen causes soreness.

Alcohol fermentation has a by product of CO2 and ethanol which is used to make alcoholic beverages. Yeast and fungus go through alcohol fermentation.

The release of CO2 by yeast is what causes bread to rise.

Alcohol Fermentation pyruvate is

converted to

ethanol in two

steps.

Alcohol

fermentation

by yeast is

used in

brewing and

winemaking.

Lactic Acid Fermentation pyruvate is reduced directly

by NADH to form lactate

Lactic acid fermentation by

some fungi and bacteria is

used to make cheese and

yogurt

The waste product,

lactate, may cause

muscle fatigue, but

ultimately it is

converted back to

pyruvate in the liver.

The Tricarboxylic Acid (TCA) Cycle

Also known as the Krebs Cycle and Citric Acid

Cycle

The citric acid cycle is the final common

pathway for the oxidation of fuel molecules:

amino acids, fatty acids, & carbohydrates.

Most fuel molecules enter the cycle as acetyl

coenzyme A

This cycle is the central metabolic hub of the

cell

The Tricarboxylic Acid (TCA) Cycle

The citric acid cycle oxidizes two-carbon units

Entry to the cycle and metabolism through it

are controlled

It is the gateway to aerobic metabolism for any

molecule that can be transformed into an

acetyl group or dicarboxylic acid,

It is also an important source of precursors for

building blocks

Overview of the TCA Cycle 1. The function of the cycle is the harvesting of high-

energy electrons from carbon fuels

2. The cycle itself neither generates ATP nor includes O2

as a reactant

3. Instead, it removes electrons from acetyl CoA & uses

them to form NADH & FADH2 (high-energy electron

carriers)

4. In oxidative phosphorylation, electrons from NADH &

FADH2 flow through a series of membrane proteins

(electron transport chain) to generate a proton gradient

Overview of the TCA Cycle

5.These protons then flow back through ATP

synthase to generate ATP from ADP & inorganic

phosphate

6.O2 is the final electron acceptor at the end of the

electron transport chain

7.The citric acid cycle + oxidative phosphorylation

provide > 95% of energy used in human aerobic

cells

Fuel for the Citric Acid Cycle

Thioester bond to acetate

-mercapto-ethylamine

Pantothenate

69

Mitochondrion

Oxidative decarboxilation of pyruvate, & citric acid cycle take place in the matrix, along with fatty acid oxidation

Site of oxidative phosphorylation

Permeable

Mitochondrion

TCA Cycle: Overview

Input: 2-carbon units

in the form of Acetyl-

CoA

Output: 2 CO2, 1 GTP,

& 8 high-energy

Electrons in the form

of reducing elements

Cellular Respiration

8 high-energy electrons from carbon fuels

Electrons reduce O2 to generate a proton gradient

ATP synthesized from proton gradient

Acetyl-CoA: Link between glycolysis and TCA

Acetyl CoA is the fuel for the citric acid cycle

Pyruvate Dehydrogenase:

AKA PDH

The enzyme that links glycolysis with other pathways

Pyruvate + CoA + NAD -> AcetylCoA + CO2 + NADH

The PDH Complex

Multi-enzyme complex Three enzymes

5 co-enzymes

Allows for efficient direct transfer of product from

one enzyme to the next

The PDH Reaction E1: pyruvate dehydrogenase

Oxidative decarboxylation of pyruvate

E2: dihydrolipoyl transacetylase Transfers acetyl group from TPP to lipoic acid

E3: dihydrolipoyl dehydrogenase Transfers acetly group to CoA, transfers electrons from reduced

lipoic acid to produce NADH

Regulation of PDH

Muscle

Resting (don’t need)

Hi energy state

Hi NADH & AcCoA

Inactivates PDH

Hi ATP & NADH & AcCoA

Inhibits PDH

Exercising (need)

Low NADH, ATP, AcCoA

Coenzymes

Vitamin B1

FAD

FAD FADH2

NAD

Step 1: Citrate formation

Enzyme: Citrate synthase

Condensation reaction Hydrolysis reaction

Dehydration Hydration

Step 2: Isomerization of citrate to isocitrate

Enzyme: Aconitase

1st NADH produced! 1st CO2 removed

Step 3: Isocitrate to α-ketoglutarate

Enzyme: Isocitrate dehydrogenase

2nd NADH produced! 2nd CO2 removed!

Step 4: Succinyl-CoA formation

Enzyme: α-ketoglutarate dehydrogenase

GTP produced • Equivalent to ATP! • GTP + ADP GDP + ATP

Step 5: Succinate formation

Enzyme: Succinyl CoA synthetase

FADH2 produced!

Step 6: Succinate to Fumarate

Enzyme: Succinate dehydrogenase

Step 7: Fumarate to Malate

Enzyme: Fumarase

3rd NADH produced

Step 8: Malate to Oxaloacetate

Enzyme: Malate dehydrogenase

The TCA Cycle

Summary of the Reactions in TCA

Regulated primarily by

ATP & NADH concentrations

control points:

Pyruvate

dehydrogenase

isocitrate

dehydrogenase

- ketoglutarate

dehydrogenase

Control of the TCA Cycle

Biosynthetic roles of the TCA cycle

OXIDATIVE PHOSPHORYLATION

2006-2007

What’s the point?

The point is to make

ATP!

ATP

ATP accounting so far…

Glycolysis 2 ATP

Kreb’s cycle 2 ATP

Life takes a lot of energy to run, need to

extract more energy than 4 ATP!

What’s the point?

A working muscle recycles over

10 million ATPs per second

There is a better way!

Electron Transport Chain

series of molecules built into inner mitochondrial

membrane

along cristae

transport proteins & enzymes

transport of electrons down ETC linked to

pumping of H+ to create H+ gradient

yields ~30-32 ATP from 1 glucose!

only in presence of O2 (aerobic respiration)

O2 That sounds more like it!

Mitochondria

Double membrane

outer membrane

inner membrane

highly folded cristae

enzymes & transport

proteins

intermembrane space

fluid-filled space between

membranes

Oooooh! Form fits function!

Electron Transport Chain

Intermembrane space

Mitochondrial matrix

Q

C

NADH dehydrogenase

cytochrome bc complex

cytochrome c oxidase complex

Inner

mitochondrial

membrane

G3P Glycolysis Krebs cycle

8 NADH 2 FADH2

Remember the Electron Carriers?

4 NADH

Time to break open the bank!

glucose

Electron Transport Chain

Intermembrane space

Mitochondrial matrix

Q

C

NADH dehydrogenase

cytochrome bc complex

cytochrome c oxidase complex

Inner

mitochondrial

membrane

But what “pulls” the electrons down the ETC?

electrons flow downhill to O2 oxidative phosphorylation!

O2

Electrons flow downhill

Electrons move in steps from

carrier to carrier downhill to O2

each carrier more electronegative

controlled oxidation

controlled release of energy

make ATP instead of fire!

H+

ADP + Pi

H+ H+

H+

H+ H+

H+ H+ H+ We did it!

Set up a H+

gradient

Allow the protons

to flow through

ATP synthase

Synthesizes ATP

ADP + Pi ATP

ATP Are we there yet?

“proton-motive” force

Chemiosmosis

The diffusion of ions across a membrane

build up of proton gradient just so H+ could flow

through ATP synthase enzyme to build ATP

Chemiosmosis links the Electron Transport Chain to ATP synthesis

So that’s the point!

Peter Mitchell

Proposed chemiosmotic hypothesis

revolutionary idea at the time

1920-1992

proton motive force

True story.

H+

H+

O2 +

Q C

32 ATP 2

Pyruvate from

cytoplasm

Electron

transport

system

ATP synthase

H2O

CO2

Krebs

cycle

Intermembrane

space Inner

mitochondrial

membrane

1. Electrons are harvested and carried to the transport system.

2. Electrons provide energy to pump protons across the membrane.

3. Oxygen joins with protons to form water.

2H+

NADH

NADH

Acetyl-CoA

FADH2

ATP

4. Protons diffuse back in down their concentration gradient, driving the synthesis of ATP.

Mitochondrial

matrix

2 1

H+

H+

O2

H+

e-

e-

e-

e-

Cellular respiration

2 ATP ~2 ATP 2 ATP ~34 ATP + + +

Pathway Substrate-Level

Phosphorylation

Oxidative

Phosphorylation

Total

ATP

Glycolysis 2 ATP 2 NADH = 4 - 6

ATP 6 - 8

CoA 2 NADH = 6 ATP 6

Krebs Cycle 2 ATP

6 NADH = 18

ATP

2 FADH2 = 4 ATP

24

TOTAL 4 ATP 32 ATP 36 - 38

Cellular respiration

Summary of cellular respiration

(carbohydrate metabolism)

Oxidative phosphorylation is the process

of making ATP from the reducing elements

NADH and FADH2, with the help of O2 and

the electron transport chain

The electron transport chain is the

structural complex that enables oxidative

phosphorylation to take place

Summary of cellular respiration

Where did the glucose come from?

Where did the O2 come from?

Where did the CO2 come from?

Where did the CO2 go?

Where did the H2O come from?

Where did the ATP come from?

What else is produced that is not listed in this equation?

Why do we breathe?

C6H12O6 6O2 6CO2 6H2O ~40 ATP + + +

ETC backs up

nothing to pull electrons down chain

NADH & FADH2 can’t unload H

ATP production ceases

cells run out of energy

and you die!

Taking it beyond…

What is the final electron acceptor in

Electron Transport Chain?

O2

So what happens if O2 unavailable?

WHOA!