Chapter 8Metabolism & Enzymes

From food webs to the life of a cell

energy

energy

energy

Flow of energy through life• Life is built on chemical reactions

– transforming energy from one form to another

organic molecules ATP & organic molecules

organic molecules ATP & organic molecules

sun

solar energy ATP & organic molecules

Concept 8.1: An organism’s metabolism transforms matter and energy, subject to the

laws of thermodynamics

• Metabolism is the totality of an organism’s chemical reactions

• Metabolism is an emergent property of life that arises from interactions between molecules within the cell

Enzyme 1

A B

Reaction 1

Enzyme 2

C

Reaction 2

Enzyme 3

D

Reaction 3

ProductStarting

molecule

Metabolism• Chemical reactions of life

– forming bonds between molecules• dehydration synthesis• synthesis• anabolic reactions

– breaking bonds between molecules• hydrolysis• digestion• catabolic reactions

That’s why they’re called

anabolic steroids!

Examples • dehydration synthesis (synthesis)

• hydrolysis (digestion)

+

H2O

+

H2O

enzyme

enzyme

Examples • dehydration synthesis (synthesis)

• hydrolysis (digestion)

enzyme

enzyme

Forms of Energy

• Energy is the capacity to cause change• Energy exists in various forms, some of which

can perform work– Kinetic energy is energy associated with motion

• Heat (thermal energy) is kinetic energy associated with random movement of atoms or molecules

– Potential energy is energy that matter possesses because of its location or structure

• Chemical energy is potential energy available for release in a chemical reaction

• Energy can be converted from one form to another

On the platform,the diver hasmore potentialenergy.

Diving convertspotentialenergy to kinetic energy.

Climbing up convertskinetic energy ofmuscle movement topotential energy.

In the water, the diver has lesspotential energy.

The Laws of Energy Transformation

• Thermodynamics is the study of energy transformations

• A closed system, such as that approximated by liquid in a thermos, is isolated from its surroundings

• In an open system, energy and matter can be transferred between the system and its surroundings

• Organisms are open systems

The First Law of Thermodynamics

• According to the first law of thermodynamics, the energy of the universe is constant– Energy can be transferred and transformed– Energy cannot be created or destroyed

• The first law is also called the principle of conservation of energy

The Second Law of Thermodynamics

• During every energy transfer or transformation, some energy is unusable, often lost as heat

• According to the second law of thermodynamics, every energy transfer or transformation increases the entropy (disorder) of the universe

LE 8-3

Chemical energy

Heat CO2

First law of thermodynamics Second law of thermodynamics

H2O

• Living cells unavoidably convert organized forms of energy to heat

• Spontaneous processes occur without energy input; they can happen quickly or slowly

• For a process to occur without energy input, it must increase the entropy of the universe

Biological Order and Disorder• Cells create ordered structures from less

ordered materials

• Organisms also replace ordered forms of matter and energy with less ordered forms

• The evolution of more complex organisms does not violate the second law of thermodynamics

• Entropy (disorder) may decrease in an organism, but the universe’s total entropy increases

Concept 8.2: The free-energy change of a reaction tells us whether the reaction occurs

spontaneously

• Biologists want to know which reactions occur spontaneously and which require input of energy

• To do so, they need to determine energy changes that occur in chemical reactions

Free-Energy Change, G

• A living system’s free energy is energy that can do work when temperature and pressure are uniform, as in a living cell

• The change in free energy (∆G) during a process is related to the change in enthalpy, or change in total energy (∆H), and change in entropy (T∆S):

∆G = ∆H - T∆S• Only processes with a negative ∆G are

spontaneous• Spontaneous processes can be harnessed to

perform work

Free Energy, Stability, and Equilibrium

• Free energy is a measure of a system’s instability, its tendency to change to a more stable state

• During a spontaneous change, free energy decreases and the stability of a system increases

• Equilibrium is a state of maximum stability• A process is spontaneous and can perform work

only when it is moving toward equilibrium

LE 8-5

Gravitational motion Diffusion Chemical reaction

Free Energy and Metabolism

• The concept of free energy can be applied to the chemistry of life’s processes

Chemical reactions & energy• Some chemical reactions release energy

– exergonic– digesting polymers– hydrolysis = catabolism

• Some chemical reactions require input of energy– endergonic– building polymers – dehydration synthesis = anabolism

digesting molecules= less organization=lower energy state

building molecules= more organization=higher energy state

Endergonic vs. exergonic reactionsexergonic endergonic

- energy released- digestion

- energy invested- synthesis

-G

G = change in free energy = ability to do work

+G

Energy & life• Organisms require energy to live

– where does that energy come from?

• coupling exergonic reactions (releasing energy) with

endergonic reactions (needing energy)

+ + energy

+ energy+

What drives reactions?• If reactions are “downhill”, why don’t they

just happen spontaneously?– because covalent bonds are stable bonds

Stable polymersdon’t spontaneously

digest into theirmonomers

Activation energy

• Breaking down large molecules requires an initial input of energy– activation energy

– large biomolecules are stable

– must absorb energy to break bonds

energycellulose CO2 + H2O + heat

Too much activation energy for life

• The amount of energy needed to destabilize the bonds of a molecule– moves the reaction over an “energy hill”

Not a match!That’s too much energy to expose

living cells to!

Reducing Activation energy• Catalysts

– reducing the amount of energy to start a reaction

Pheeew…that takes a lot

less energy!

Equilibrium and Metabolism• Reactions in a closed system eventually reach

equilibrium and then do no work

• Cells are not in equilibrium; they are open systems experiencing a constant flow of materials

• A catabolic pathway in a cell releases free energy in a series of reactions

• Closed and open hydroelectric systems can serve as analogies

Concept 8.3: ATP powers cellular work by coupling exergonic reactions to endergonic

reactions

• A cell does three main kinds of work:– Mechanical– Transport– Chemical

• To do work, cells manage energy resources by energy coupling, the use of an exergonic process to drive an endergonic one

The Structure and Hydrolysis of ATP

• ATP (adenosine triphosphate) is the cell’s energy shuttle

• ATP provides energy for cellular functions

LE 8-8

Phosphate groups

Ribose

Adenine

• The bonds between the phosphate groups of ATP’s tail can be broken by hydrolysis

• Energy is released from ATP when the terminal phosphate bond is broken

• This release of energy comes from the chemical change to a state of lower free energy, not from the phosphate bonds themselves

LE 8-9

Adenosine triphosphate (ATP)

Energy

P P P

PPP i

Adenosine diphosphate (ADP)Inorganic phosphate

H2O

+ +

• In the cell, the energy from the exergonic reaction of ATP hydrolysis can be used to drive an endergonic reaction

• Overall, the coupled reactions are exergonic

LE 8-10

Endergonic reaction: G is positive, reactionis not spontaneous

Exergonic reaction: G is negative, reactionis spontaneous

G = +3.4 kcal/mol

G = –7.3 kcal/mol

G = –3.9 kcal/mol

NH2

NH3Glu Glu

Glutamicacid

Coupled reactions: Overall G is negative;together, reactions are spontaneous

Ammonia Glutamine

ATP H2O ADP P i

+

+ +

How ATP Performs Work• ATP drives endergonic reactions by

phosphorylation, transferring a phosphate group to some other molecule, such as a reactant

• The recipient molecule is now phosphorylated• The three types of cellular work (mechanical,

transport, and chemical) are powered by the hydrolysis of ATP

LE 8-11

NH2

Glu

P i

P i

P i

P i

Glu NH3

P

P

P

ATPADP

Motor protein

Mechanical work: ATP phosphorylates motor proteins

Protein moved

Membraneprotein

Solute

Transport work: ATP phosphorylates transport proteins

Solute transported

Chemical work: ATP phosphorylates key reactants

Reactants: Glutamic acidand ammonia

Product (glutamine)made

+ +

+

The Regeneration of ATP• ATP is a renewable resource that is

regenerated by addition of a phosphate group to ADP

• The energy to phosphorylate ADP comes from catabolic reactions in the cell

• The chemical potential energy temporarily stored in ATP drives most cellular work

LE 8-12

Pi

ADP

Energy for cellular work

(endergonic, energy-

consuming processes)

Energy from catabolism

(energonic, energy-

yielding processes)

ATP

+

Concept 8.4: Enzymes speed up metabolic reactions by lowering energy barriers

• A catalyst is a chemical agent that speeds up a reaction without being consumed by the reaction

• An enzyme is a catalytic protein• Hydrolysis of sucrose by the enzyme sucrase is

an example of an enzyme-catalyzed reaction

LE 8-13

SucroseC12H22O11

GlucoseC6H12O6

FructoseC6H12O6

The Activation Energy Barrier• Every chemical reaction between molecules

involves bond breaking and bond forming

• The initial energy needed to start a chemical reaction is called the free energy of activation, or activation energy (EA)

• Activation energy is often supplied in the form of heat from the surroundings

LE 8-14

Transition state

C D

A B

EA

Products

C D

A B

G < O

Progress of the reaction

Reactants

C D

A B

Fre

e en

erg

y

How Enzymes Lower the EA Barrier

• Enzymes catalyze reactions by lowering the EA barrier

• Enzymes do not affect the change in free-energy (∆G); instead, they hasten reactions that would occur eventually

Animation: How Enzymes Work

LE 8-15

Course ofreactionwithoutenzyme

EA

without enzyme

G is unaffectedby enzyme

Progress of the reaction

Fre

e en

erg

y

EA withenzymeis lower

Course ofreactionwith enzyme

Reactants

Products

Substrate Specificity of Enzymes

• The reactant that an enzyme acts on is called the enzyme’s substrate

• The enzyme binds to its substrate, forming an enzyme-substrate complex

• The active site is the region on the enzyme where the substrate binds

• Induced fit of a substrate brings chemical groups of the active site into positions that enhance their ability to catalyze the reaction

LE 8-16

Substrate

Active site

Enzyme Enzyme-substratecomplex

Catalysis in the Enzyme’s Active Site

• In an enzymatic reaction, the substrate binds to the active site

• The active site can lower an EA barrier by

– Orienting substrates correctly– Straining substrate bonds– Providing a favorable microenvironment– Covalently bonding to the substrate

LE 8-17

Enzyme-substratecomplex

Substrates

Enzyme

Products

Substrates enter active site; enzymechanges shape so its active siteembraces the substrates (induced fit).

Substrates held inactive site by weakinteractions, such ashydrogen bonds andionic bonds.

Active site (and R groups ofits amino acids) can lower EA

and speed up a reaction by• acting as a template for substrate orientation,• stressing the substrates and stabilizing the transition state,• providing a favorable microenvironment,• participating directly in the catalytic reaction.

Substrates areconverted intoproducts.

Products arereleased.

Activesite is

availablefor two new

substratemolecules.

Effects of Local Conditions on Enzyme Activity

• An enzyme’s activity can be affected by:– General environmental factors, such as

temperature and pH– Chemicals that specifically influence the

enzyme

Effects of Temperature and pH

• Each enzyme has an optimal temperature in which it can function

• Each enzyme has an optimal pH in which it can function

LE 8-18

Optimal temperature fortypical human enzyme

Optimal temperature forenzyme of thermophilic (heat-tolerant bacteria)

Temperature (°C)

Optimal temperature for two enzymes

0 20 40 60 80 100

Ra

te o

f re

ac

tio

n

Optimal pH for pepsin(stomach enzyme)

Optimal pHfor trypsin(intestinalenzyme)

pH

Optimal pH for two enzymes

0

Ra

te o

f re

ac

tio

n

1 2 3 4 5 6 7 8 9 10

Cofactors

• Cofactors are nonprotein enzyme helpers

• Coenzymes are organic cofactors

Enzyme Inhibitors• Competitive inhibitors bind to the active site of

an enzyme, competing with the substrate

• Noncompetitive inhibitors bind to another part of an enzyme, causing the enzyme to change shape and making the active site less effective

LE 8-19Substrate

Active site

Enzyme

Competitiveinhibitor

Normal binding

Competitive inhibition

Noncompetitive inhibitor

Noncompetitive inhibition

A substrate canbind normally to the

active site of anenzyme.

A competitiveinhibitor mimics the

substrate, competingfor the active site.

A noncompetitiveinhibitor binds to the

enzyme away from theactive site, altering the

conformation of theenzyme so that its

active site no longerfunctions.

Concept 8.5: Regulation of enzyme activity helps control metabolism

• Chemical chaos would result if a cell’s metabolic pathways were not tightly regulated

• To regulate metabolic pathways, the cell switches on or off the genes that encode specific enzymes

Allosteric Regulation of Enzymes

• Allosteric regulation is the term used to describe cases where a protein’s function at one site is affected by binding of a regulatory molecule at another site

• Allosteric regulation may either inhibit or stimulate an enzyme’s activity

Allosteric Activation and Inhibition

• Most allosterically regulated enzymes are made from polypeptide subunits

• Each enzyme has active and inactive forms• The binding of an activator stabilizes the active

form of the enzyme• The binding of an inhibitor stabilizes the

inactive form of the enzyme

LE 8-20a

Allosteric enzymewith four subunits

Regulatorysite (oneof four) Active form

Activator

Stabilized active form

Active site(one of four)

Allosteric activatorstabilizes active form.

Non-functionalactive site

Inactive formInhibitor

Stabilized inactive form

Allosteric inhibitorstabilizes inactive form.

Oscillation

Allosteric activators and inhibitors

• Cooperativity is a form of allosteric regulation that can amplify enzyme activity

• In cooperativity, binding by a substrate to one active site stabilizes favorable conformational changes at all other subunits

LE 8-20b

Substrate

Binding of one substrate molecule toactive site of one subunit locks allsubunits in active conformation.

Cooperativity another type of allosteric activation

Stabilized active formInactive form

Feedback Inhibition

• In feedback inhibition, the end product of a metabolic pathway shuts down the pathway

• Feedback inhibition prevents a cell from wasting chemical resources by synthesizing more product than is needed

LE 8-21

Active siteavailable

Initial substrate(threonine)

Threoninein active site

Enzyme 1(threoninedeaminase)

Enzyme 2

Intermediate A

Isoleucineused up bycell

Feedbackinhibition Active site of

enzyme 1 can’tbindtheoninepathway off

Isoleucinebinds toallostericsite

Enzyme 3

Intermediate B

Enzyme 4

Intermediate C

Enzyme 5

Intermediate D

End product(isoleucine)

Specific Localization of Enzymes Within the Cell

• Structures within the cell help bring order to metabolic pathways

• Some enzymes act as structural components of membranes

• Some enzymes reside in specific organelles, such as enzymes for cellular respiration being located in mitochondria

LE 8-22

Mitochondria,sites of cellular respiration

1 µm

Catalysts

• So what’s a cell got to do to reduce activation energy?– get help! … chemical help…

ENZYMES

G

Call in the ENZYMES!

Enzymes • Biological catalysts

– proteins (& RNA) – facilitate chemical reactions

• increase rate of reaction without being consumed• reduce activation energy• don’t change free energy (G) released or required

– required for most biological reactions– highly specific

• thousands of different enzymes in cells

– control reactionsof life

Enzymes vocabularysubstrate

• reactant which binds to enzyme• enzyme-substrate complex: temporary association

product • end result of reaction

active site • enzyme’s catalytic site; substrate fits into active site

substrate

enzyme

productsactive site

Properties of enzymes• Reaction specific

– each enzyme works with a specific substrate • chemical fit between active site & substrate

– H bonds & ionic bonds

• Not consumed in reaction– single enzyme molecule can catalyze thousands

or more reactions per second• enzymes unaffected by the reaction

• Affected by cellular conditions– any condition that affects protein structure

• temperature, pH, salinity

Naming conventions• Enzymes named for reaction they catalyze

– sucrase breaks down sucrose

– proteases break down proteins

– lipases break down lipids

– DNA polymerase builds DNA• adds nucleotides

to DNA strand

– pepsin breaks down proteins (polypeptides)

Lock and Key model• Simplistic model of

enzyme action– substrate fits into 3-D

structure of enzyme’ active site

• H bonds between substrate & enzyme

– like “key fits into lock”

Size doesn’t matter…Shape matters!

Induced fit model• More accurate model of enzyme action

– 3-D structure of enzyme fits substrate– substrate binding cause enzyme to change

shape leading to a tighter fit • “conformational change”• bring chemical groups in position to catalyze

reaction

How does it work?• Variety of mechanisms to lower

activation energy & speed up reaction– synthesis

• active site orients substrates in correct position for reaction

– enzyme brings substrate closer together

– diegstion• active site binds substrate & puts stress on

bonds that must be broken, making it easier to separate molecules

Got any Questions?!

Factors that Affect Enzymes

Factors Affecting Enzyme Function• Enzyme concentration

• Substrate concentration• Temperature • pH• Salinity• Activators• Inhibitors

catalase

Enzyme concentration

enzyme concentration

reac

tio

n r

ate

What’shappening here?!

Factors affecting enzyme function

• Enzyme concentration – as enzyme = reaction rate

• more enzymes = more frequently collide with substrate

– reaction rate levels off• substrate becomes limiting factor• not all enzyme molecules can find substrate

enzyme concentration

reac

tio

n r

ate

Substrate concentration

substrate concentration

reac

tio

n r

ate

What’shappening here?!

Factors affecting enzyme function

substrate concentration

reac

tio

n r

ate

• Substrate concentration – as substrate = reaction rate

• more substrate = more frequently collide with enzyme

– reaction rate levels off• all enzymes have active site engaged• enzyme is saturated• maximum rate of reaction

37°

Temperature

temperature

reac

tio

n r

ate

What’shappening here?!

Factors affecting enzyme function

• Temperature– Optimum T°

• greatest number of molecular collisions• human enzymes = 35°- 40°C

– body temp = 37°C

– Heat: increase beyond optimum T°• increased energy level of molecules disrupts bonds in

enzyme & between enzyme & substrate– H, ionic = weak bonds

• denaturation = lose 3D shape (3° structure)– Cold: decrease T°

• molecules move slower • decrease collisions between enzyme & substrate

Enzymes and temperature

• Different enzymes function in different organisms in different environments

37°Ctemperature

reac

tio

n r

ate

70°C

human enzymehot spring

bacteria enzyme

(158°F)

How do ectotherms do it?

7

pH

pH

reac

tio

n r

ate

20 1 3 4 5 6 8 9 10

pepsin trypsin

What’shappening here?!

11 12 13 14

pepsin

trypsin

Factors affecting enzyme function• pH

– changes in pH• adds or remove H+

• disrupts bonds, disrupts 3D shape – disrupts attractions between charged amino acids – affect 2° & 3° structure– denatures protein

– optimal pH?• most human enzymes = pH 6-8

– depends on localized conditions– pepsin (stomach) = pH 2-3– trypsin (small intestines) = pH 8

720 1 3 4 5 6 8 9 10 11

Salinity

salt concentration

reac

tio

n r

ate

What’shappening here?!

Factors affecting enzyme function• Salt concentration

– changes in salinity• adds or removes cations (+) & anions (–)• disrupts bonds, disrupts 3D shape

– disrupts attractions between charged amino acids – affect 2° & 3° structure– denatures protein

– enzymes intolerant of extreme salinity• Dead Sea is called dead for a reason!

Compounds which help enzymes

• Activators– cofactors

• non-protein, small inorganic compounds & ions– Mg, K, Ca, Zn, Fe, Cu– bound within enzyme molecule

– coenzymes• non-protein, organic molecules

– bind temporarily or permanently toenzyme near active site

• many vitamins– NAD (niacin; B3)– FAD (riboflavin; B2)– Coenzyme A

Mg inchlorophyll

Fe inhemoglobin

Compounds which regulate enzymes• Inhibitors

– molecules that reduce enzyme activity– competitive inhibition– noncompetitive inhibition– irreversible inhibition– feedback inhibition

Competitive Inhibitor • Inhibitor & substrate “compete” for active site

– penicillin blocks enzyme bacteria use to build cell walls

– disulfiram (Antabuse)treats chronic alcoholism

• blocks enzyme that breaks down alcohol

• severe hangover & vomiting5-10 minutes after drinking

• Overcome by increasing substrate concentration– saturate solution with substrate

so it out-competes inhibitor for active site on enzyme

Non-Competitive Inhibitor • Inhibitor binds to site other than active site

– allosteric inhibitor binds to allosteric site – causes enzyme to change shape

• conformational change• active site is no longer functional binding site

– keeps enzyme inactive

– some anti-cancer drugsinhibit enzymes involved in DNA synthesis

• stop DNA production

• stop division of more cancer cells

– cyanide poisoningirreversible inhibitor of Cytochrome C, an enzyme in cellular respiration

• stops production of ATP

Irreversible inhibition• Inhibitor permanently binds to enzyme

– competitor• permanently binds to active site

– allosteric• permanently binds to allosteric site • permanently changes shape of enzyme• nerve gas, sarin, many insecticides (malathion,

parathion…)– cholinesterase inhibitors

» doesn’t breakdown the neurotransmitter, acetylcholine

Allosteric regulation• Conformational changes by regulatory

molecules – inhibitors

• keeps enzyme in inactive form

– activators • keeps enzyme in active form

Conformational changes Allosteric regulation

Metabolic pathways

A B C D E F G enzyme

1

enzyme

2

enzyme3

enzyme4

enzyme5

enzyme6

• Chemical reactions of life are organized in pathways– divide chemical reaction into

many small steps• artifact of evolution efficiency

– intermediate branching points control = regulation

A B C D E F G enzyme

Efficiency• Organized groups of enzymes

– enzymes are embedded in membrane and arranged sequentially

• Link endergonic & exergonic reactionsWhoa!

All that going onin those littlemitochondria!

allosteric inhibitor of enzyme 1

Feedback Inhibition• Regulation & coordination of production

– product is used by next step in pathway– final product is inhibitor of earlier step

• allosteric inhibitor of earlier enzyme• feedback inhibition

– no unnecessary accumulation of product

A B C D E F G enzyme

1

enzyme2

enzyme3

enzyme4

enzyme

5

enzyme6

X

Feedback inhibition• Example

– synthesis of amino acid, isoleucine from amino acid, threonine

– isoleucine becomes the allosteric inhibitor of the first step in the pathway

• as product accumulates it collides with enzyme more often than substrate does

threonine

isoleucine

Don’t be inhibited! Ask Questions!

Cooperativity

• Substrate acts as an activator– substrate causes conformational

change in enzyme• induced fit

– favors binding of substrate at 2nd site– makes enzyme more active & effective

• hemoglobinHemoglobin 4 polypeptide chains can bind 4 O2; 1st O2 binds now easier for other

3 O2 to bind