Introduction to Metabolism

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 diversityAutotrophs 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 PathwaysPathways consist of sequential steps

The enzymes may be: Separate Form a multienzyme complexA 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 light energy of ATP In heterotrophs, catabolism produces

ATP, which drives activities of cellsATP cycle carries energy from

photosynthesis or catabolism to the energy-requiring processes of cells

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

WHY ATP?

Free energy is released when ATP is hydrolyzed.

This energy drives reactions that need it (eg. muscle contraction)

Recall coupled reactionsATP has a higher phosphoryl

transfer potential

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 bondsUses 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 glucoseProducts:

2 Pyruvate2 ATP2 NADH

Cytosolic

Glycolysis

GlycolysisAnaerobic

The entire process does not require O2

Glycolysis: General FunctionsProvide energy in the form of ATPGenerate intermediates for other

pathways:Hexose monophosphate pathwayGlycogen synthesisPyruvate 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 exerciseAdipose 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 concentrationMuscle & Adipose:

The GLUT-4 transporter is sensitive to insulin

Glucose Utilization

Phosphorylation of glucoseCommits glucose for use by that cellEnergy consuming

Hexokinase: muscle and other tissues

Glucokinase: liver

Properties of Glucokinase and Hexokinase

Regulation of Cellular Glucose Utilization in the LiverFeeding

Blood glucose concentration high GLUT-2 taking up glucose Glucokinase induced by insulin High cell glucose allows GK to phosphorylate

glucose for use by liverPost-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

StarvationBlood & cell glucose concentration lowGLUT-2 not taking up glucoseGK synthesis repressedGlucose 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 utilizationStarving 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 HKHigh glycolysis from glycogen or

blood glucose

Regulation of Glycolysis

Regulation of 3 irreversible stepsPFK-1 is rate limiting enzyme and

primary site of regulation.

Regulation of Glycolysis

Most important regulation hub!

Regulation of PFK-1 in Muscle

Relatively constitutiveAllosterically stimulated by AMP

High glycolysis during exerciseAllosterically 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 insulinRepressed in starvation by glucagon

Allosteric regulationLike muscle w/ AMP, ATP, CitrateActivated by Fructose-2,6-

bisphosphate

FermentationAnaerobic respiration!Produces ATP without oxygen.No ETC is present since there is no

oxygenNAD+ gets recycled by use of an

organic hydrogen acceptor like lactate or ethanol.

Common in prokaryotes and very useful to humans.

FermentationTwo 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 Fermentationpyruvate 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 CycleThe citric acid cycle is the final common

pathway for the oxidationof 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

unitsEntry 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 Cycle1. The function of the cycle is the harvesting of

high-energy electrons from carbon fuels2. The cycle itself neither generates ATP nor

includes O2 as a reactant3. Instead, it removes electrons from acetyl CoA &

uses them to form NADH & FADH2 (high-energy electron carriers)

4. In oxidative phosphorylation, electrons from reoxidation of NADH & FADH2 flow through a series of membrane proteins (electron transport chain) to generate a proton gradient

Overview of the TCA Cycle5.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 cytric acid cycle + oxidative phosphorylation provide > 95% of energy used in human aerobic cells

Fuel for the Citric Acid Cycle

Thioester bondto acetate

-mercapto-ethylamine

Pantothenate

70

Mitochondrion

Double membrane, & cristae: invaginations of inner membrane

Oxidative decarboxilationof pyruvate, & citric acidcycle take place in the matrix, along with fatty acid oxidationSite 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-energyElectrons in the form of reducing elements

Cellular Respiration

8 high-energyelectrons fromcarbon fuels

Electrons reduceO2 to generate aproton gradient

ATP synthesizedfrom protongradient

Acetyl-CoA: Link between glycolysis and TCA

Acetyl CoA is thefuel for the citric acidcycle

Pyruvate Dehydrogenase: AKA PDH The enzyme that links glycolysis with other

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

NADH

The PDH ComplexMulti-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 PDHMuscle

Resting (don’t need) Hi energy state Hi NADH & AcCoA

Inactivates PDH Hi ATP & NADH & AcCoA

Inhibits PDHExercising (need)

Low NADH, ATP, AcCoA

Regulation of PDHLiver

Fed (need to make FA)Hi energy Insulin activates

PDHStarved (don’t

need)Hi energyNo insulin

PDH inactive

Coenzymes

Vitamin B1

FAD

FAD FADH2

NAD

Step 1: Citrate formationEnzyme: 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-ketoglutarateEnzyme: Isocitrate dehydrogenase

2nd NADH produced! 2nd CO2 removed!

Step 4: Succinyl-CoA formationEnzyme: α-ketoglutarate dehydrogenase

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

Step 5: Succinate formationEnzyme: Succinyl CoA synthetase

FADH2 produced!

Step 6: Succinate to FumarateEnzyme: Succinate dehydrogenase

Step 7: Fumarate to MalateEnzyme: Fumarase

3rd NADH produced

Step 8: Malate to OxaloacetateEnzyme: Malate dehydrogenase

The TCA Cycle

Summary of the Reactions in TCA

Regulated primarily byATP & 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 thepoint?

The pointis 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) O2Thatsounds morelike 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 ChainIntermembrane space

Mitochondrial matrix

Q

C

NADH dehydrogenase

cytochromebc complex

cytochrome coxidase complex

Innermitochondrialmembrane

G3P

Glycolysis

Krebs cycle

8 NADH2 FADH2

Remember the Electron Carriers?

4 NADH

Time tobreak openthe bank!

glucose

Electron Transport ChainIntermembrane space

Mitochondrial matrix

Q

C

NADH dehydrogenase

cytochromebc complex

cytochrome coxidase complex

Innermitochondrialmembrane

But what “pulls” the electrons down the ETC?

electronsflow downhill to O2 oxidative phosphorylation!

O2

Electrons flow downhillElectrons move in steps from

carrier to carrier downhill to O2 each carrier more electronegative controlled oxidation controlled release of energy

make ATPinstead offire!

H+

ADP + Pi

H+ H+

H+

H+ H+

H+H+H+We did it!

ATP

Set up a H+

gradientAllow the

protons to flow through ATP synthase

Synthesizes ATP

ADP + Pi ATPAre wethere yet?

“proton-motive” force

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

Chemiosmosis links the Electron Transport Chain to ATP synthesis

So that’sthe point!

Peter MitchellProposed chemiosmotic

hypothesis revolutionary idea at the time

1920-1992

proton motive force

True story.

H+

H+

O2+

Q C

32ATP2

Pyruvate fromcytoplasm

Electrontransportsystem

ATPsynthase

H2O

CO2

Krebscycle

IntermembranespaceInner

mitochondrialmembrane

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

ATP4. Protons diffuse back in down their concentration gradient, driving the synthesis of ATP.

Mitochondrial matrix

21

H+

H+

O2

H+

e-

e-

e-

e-

Cellular respiration

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

~40 ATP

Pathway Substrate-LevelPhosphorylation

OxidativePhosphorylation

TotalATP

Glycolysis 2 ATP 2 NADH = 4 - 6 ATP 6 - 8

CoA   2 NADH = 6 ATP 6

Krebs Cycle 2 ATP6 NADH = 18 ATP2 FADH2 = 4 ATP

24

TOTAL 4 ATP 32 ATP 36 - 38

Cellular respiration36-38 ATP

Summary of cellular respirationOxidative 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!