chapter 6 lecture slides - health science...

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CHAPTER 6

LECTURE

SLIDES

Prepared by

Brenda Leady University of Toledo

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2

Energy

Ability to promote change or do work

2 forms

Kinetic- associated with movement

Potential- due to structure or location

Chemical energy- energy in molecular bonds

4

2 Laws of thermodynamics

1. First law

Law of conservation of energy

Energy cannot be created or destroyed

Can be transformed from one type to another

2. Second law

Transfer or transformation of energy from

one form to another increases entropy or

degree of disorder of a system

5


Highly

ordered

6


Increase

More disordered

in entropy

Highly

ordered

7

Change in free energy determines direction

Total energy = Usable energy + Unusable energy

Energy transformations involve an increase

in entropy

Entropy - a measure of the disorder that

cannot be harnessed to do work

8

H = G + TS

H= enthalpy or total energy

G= free energy or amount of energy

for work

S= entropy or unusable energy

T= absolute temperature in Kelvin (K)

9

Spontaneous reactions?

Occur without input of additional energy

Not necessarily fast

Key factor is the free energy change

10

ΔG = Gproducts - Greactants

Exergonic

ΔG<0 or negative free energy change

Spontaneous

Endergonic

ΔG>0 or positive free energy change

Requires addition of free energy

Not spontaneous

11

Hydrolysis of ATP

ΔG = -7.3 kcal/mole

Reaction favors

formation of

products

Energy liberated can

drive a variety of

cellular processes


Adenine (A)

Ribose

Phosphate groups

Phosphate (Pi) Adenosine diphosphate (ADP)

Adenosine triphosphate (ATP)

+ ~

H H

OH

O

OH

H H

O O

O

H2C

O–

NH2

N

N

H

N

N

P OH

O

O–

P O–

O

O–

P HO

~ ~

H H

OH

O

OH

H H

O O

O

H2C

O–

NH2

N

N

H

N

N

P O

O

O–

P

O

O–

P O–

H2O Hydrolysis

of ATP

12

Cells use ATP hydrolysis

An endergonic reaction can be coupled to

an exergonic reaction

Endergonic reaction will be spontaneous if

net free energy change for both processes

is negative

13

Glucose + phosphate → glucose-phosphate + H2O

ΔG = +3.3 Kcal/mole

endergonic

ATP + H2O → ADP + Pi

ΔG = -7.3 Kcal/mole

exergonic

Coupled reaction: Glucose + ATP → glucose-phosphate + ADP

ΔG = -4.0 Kcal/mole

exergonic

14

Enzymes and Ribozymes

A spontaneous reaction is not necessarily

a fast reaction

Catalyst- agent that speeds up the rate of

a chemical reaction without being

consumed during the reaction

Enzymes- protein catalysts in living cells

Ribozymes- RNA molecules with catalytic

properties

15

Activation energy

Initial input of energy to start reaction

Allows molecules to get close enough to

cause bond rearrangement

Can now achieve transition state where

bonds are stretched

16

Overcoming activation energy

2 common ways

Large amounts of heat

Using enzymes to lower activation energy

Small amount of heat can now push reactants to

transition state

17


Progress of an exergonic reaction

Fre

e e

nerg

y (

G)

Transition state

Reactants

Reactant molecules

Enzyme

ATP

Glucose

Products

Activation energy (EA)

without enzyme

Activation energy (EA)

with enzyme

Change

in free

energy

(G)

18

Lowering activation energy

Straining bonds in reactants to make it

easier to achieve transition state

Positioning reactants together to facilitate

bonding

Changing local environment

Direct participation through very temporary

bonding

19

Other enzyme features

Active site- location where reaction takes

place

Substrate- reactants that bind to active site

Enzyme-substrate complex formed when

enzyme and substrate bind

20


Glucose

Substrates

Active site ATP

21


Glucose Substrates

Active site ATP

Enzyme-substrate complex

22

Glucose Substrates

Active site ATP



23


Glucose Substrates

Active site ATP


Glucose-

phosphate

ADP

24

Substrate binding

Enzymes have a high affinity or high

degree of specificity for a substrate

Example of a lock and key for substrate

and enzyme binding

Induced fit- interaction also involves

conformational changes

Enzyme reactions

Saturation- plateau where nearly all active sites

occupied by substrate

Vmax = velocity of reaction near maximal rate

Km = substrate concentration at which velocity is

half maximal value

Also called Michaelis constant

High Km enzyme needs higher substrate

concentration

Inversely related to affinity between enzyme and

substrate

25

26

Velo

cit

y

(pro

du

ct/

seco

nd

)

[Substrate]

A

B

Vmax

2

Vmax C

D

A

60 sec

Low

B

60 sec

Moderate

C

60 sec

High

D

60 sec

(a) Reaction velocity in the absence of inhibitors

0

Amount of

enzyme

Tube

Incubation

time

Substrate

concentration

Very

high

1 m 1 m 1 m 1 m

KM


Inhibition

Competitive inhibition

Molecule binds to active site

Inhibits ability of substrate to bind

Apparent Km increases- more substrate

needed

Noncompetitive

Lowers Vmax without affecting Km

Inhibitor binds to allosteric site- not active site

27

28

KM with inhibitor [Substrate]

(b) Competitive inhibition

Ve

loc

ity

(pro

du

ct/

se

co

nd

)

Plus competitive inhibitor

Substrate

Inhibitor

Enzyme

Vmax

KM [Substrate]

(c) Noncompetitive inhibition

Ve

loc

ity

(pro

du

ct/

se

co

nd

)

Substrate

Inhibitor

Enzyme

Allosteric site

0

0

KM

Vmax

V max with inhibitor

Plus noncompetitive

inhibitor


29

Other requirements for enzymes

Prosthetic groups- small molecules

permanently attached to the enzyme

Cofactor- usually inorganic ion that

temporarily binds to enzyme

Coenzyme- organic molecule that

participates in reaction but left unchanged

afterward

30

Enzymes are affected by environment

Most enzymes function maximally in a

narrow range of temperature and pH

Outside of this narrow range, enzyme

function decreases

31

0 0

10 20

Rate

of

a

ch

em

ical

reac

tio

n

30 40 50 60

High

Temperature (ºC)


Discovery of Ribozymes

Until 1980s, scientists thought all biological catalysts were proteins

Ribonuclease P (Rnase P) found in all living organisms

Involved in processing tRNA molecules

Ribonucleoprotein- 1 RNA and 1 protein subunit

Experiments found RNA subunit alone was able to cleave substrate

True catalyst- accelerates rate without being altered


ptRNA

tRNA

ptRNA

ptRNA

tRNA

Experimental level Conceptual level

MgCl2

Low MgCl2

(10 mM)

High MgCl2

(100 mM)

RNA

subunit

alone

RNA subunit

plus protein

subunit

Higher

mass

Lower

mass

3´

5´

RNA subunit

alone cuts here

3´

5´

5´ fragment

5´ +

5´ fragment

Catalytic function will

result in the digestion

of ptRNA into tRNA and

a smaller 5´ fragment .

5´ fragment

ptRNA

3´

5´

1 2 3 4 5

THE DATA 5

© Altman, S., (1990). Nobel Lecture: Enzymatic Cleavage of RNA by RNA. Bioscience Reports, 10, 317–337. Fig. 7

34

Overview of metabolism

Chemical reactions occur in metabolic

pathways

Each step is coordinated by a specific

enzyme

Catabolic pathways

Result in breakdown and are exergonic

Anabolic pathways

Promote synthesis and are endergonic

Must be coupled to exergonic reaction

35

Initial substrate

OH

OH OH


36

PO42—

Enzyme 1

Initial substrate Intermediate 1

OH

OH OH OH OH


37

Enzyme 1

Initial substrate Intermediate 1 Intermediate 2

Enzyme 2

OH

OH OH OH OH OH


PO42— PO4

2—

PO42—

38

Enzyme 1

Initial substrate Intermediate 1 Intermediate 2 Final product

Enzyme 2 Enzyme 3

OH

OH OH OH OH OH


PO42— PO4

2— PO42—

PO42— PO4

2— PO42—

39

Catabolic reactions

Breakdown of reactants

Used for recycling

Used to obtain energy for endergonic

reactions

Energy stored in energy intermediates

ATP, NADH

40

2 ways to make ATP

1. Substrate-level phosphorylation

Enzyme directly transfers phosphate from

one molecule to another molecule

2. Chemiosmosis

Energy stored in an electrochemical gradient

is used to make ATP from ADP and Pi

41

Redox

Oxidation

Removal of electrons

Reduction

Addition of electrons

Redox reaction

Electron removed from one molecule is added

to another

42

Ae- + B → A + Be-

A

Has been oxidized

Electron removed

B

Has been reduced

Electron added

43

Energy intermediates

Electrons removed by oxidation are used

to create energy intermediates like NADH

NAD+ Nicotinamide adenine dinucleotide

NADH…

Oxidized to make ATP

Can donate electrons during synthesis

reactions

44

Adenine

Nicotinamide

Nicotinamide

adenine

dinucleotide

(NAD+)


+ 2e– + H+

+

45

Reduction

Oxidation

Adenine

Nicotinamide

NADH

(an electron

carrier)

Nicotinamide

adenine

dinucleotide

(NAD+)


+ 2e– + H+

+

46

Anabolic reactions

Biosynthetic reactions

Make large macromolecules or smaller

molecules not available from food

Many proteins use ATP as a

source of energy

Each ATP undergoes 10,000 cycles of hydrolysis and resynthesis every day

Particular amino acid sequences in proteins function as ATP-binding sites

Can predict whether a newly discovered protein uses ATP or not

On average, 20% of all proteins bind ATP

Likely underestimated because there may be other types of ATP-binding sites

Enormous importance of ATP as energy source

Synthesis

Hydrolysis

ADP + Pi

ATP + H2O Energy release

(Exergonic)

Energy input

(Endergonic)


49

Regulation of metabolic pathways

1. Gene regulation

Turn on or off genes

2. Cellular regulation

Cell-signaling pathways like hormones

3. Biochemical regulation

Feedback inhibition- product of pathway

inhibits early steps to prevent

overaccumulation of product

50


Enzyme 1

Initial substrate

Allosteric site

Active site

51


Enzyme 1

Initial substrate

Allosteric site

Intermediate 1

Active site

Final product

Conformational

change

52


Enzyme 1

Initial substrate

Allosteric site

Intermediate 1

Enzyme 2

Active site

Final product

Conformational

change

53

Enzyme 1

Initial substrate

Allosteric site

Intermediate 1 Intermediate 2

Enzyme 2

Active site

Final product

Conformational

change


54

Enzyme 1

Initial substrate

Allosteric site

Intermediate 1 Intermediate 2

Enzyme 2

Enzyme 3

Active site

Final product

Conformational

change


55

Enzyme 1

Initial substrate

Conformational

change

Allosteric site

Intermediate 1 Intermediate 2 Final product

Enzyme 2

Enzyme 3

Active site

Final product


56


Enzyme 1

Initial substrate

Conformational

change

Allosteric site

Intermediate 1 Intermediate 2 Final product

Enzyme 2

Enzyme 3

Active site

Final product

57

Recycling

Most large molecules exist for a relatively

short period of time

Half-life- time it takes for 50% of the

molecules to be broken down and recycled

All living organisms must efficiently use

and recycle organic molecules

Expression of genome allows cells to

respond to changes in their environment

RNA and proteins made when needed

Broken down when they are not

mRNA degradation important

Conserve energy by degrading mRNAs for

proteins no longer required

Remove faulty copies of mRNA

58

mRNA degradation

Exonucleases

Enzyme cleaves off nucleotides from end

Exosome

Multiprotein complex uses exonucleases

59

60

A Cap mRNA

Poly A tail 5´

3´


61

A Cap mRNA

Poly A tail

Poly A tail is shortened.

5´

5´

3´

3´


62

A Cap mRNA

Poly A tail


5´

5´ cap is removed.

5´ 3´

5´

3´

3´


63


A

Exonuclease

Cap mRNA

Poly A tail


5´

5´ cap is removed.

Nucleotides

are recycled.

5´ 3´

RNA is degraded in

the 5´ to 3´ direction

via an exonuclease.

5´

3´

3´

3´

64

A

Exonuclease

Cap mRNA

Poly A tail


RNA is degraded in


via the exosome.

5´

5´ cap is removed.

Nucleotides

are recycled.

5´ 3´

RNA is degraded in


via an exonuclease.

5´

3´

Nucleotides

are recycled.

3´

5´

3´ Exosome


65


A

Exonuclease

Cap

Exosome

mRNA

Poly A tail


RNA is degraded in


via the exosome.

5´

5´ cap is removed.

Nucleotides

are recycled.

5´ 3´

RNA is degraded in


via an exonuclease.

5´

3´

(a) 5´ (b) 3´ 5´ degradation by exosome

Nucleotides

are recycled.

3´ degradation by exonuclease

3´

5´

3´

© Liu, Q., Greimann, J.C., and Lima, C.D., (2006). Reconstitution, activities, and structure of the eukaryotic exosome. Cell, 127, 1223-1237.

Graphic generated using DeLano, W.L. (2002). The PyMOL Molecular Graphics System (San Carlos, CA, USA, DeLano Scientific)

Lysosomes contain hydrolases to break

down proteins, carbohydrates, nucleic

acids, and lipids

Digest substances taken up by endocytosis

Autophagy- recycling worn out organelles

Autophagosome

Proteosome digests proteins targeted for

destruction with ubiquitin.

66

67

Ubiquitin

Target

protein


68

Ubiquitin

Target

protein


69


Ubiquitin

Target

protein

70

Cap

1

2

3

4

Cap

(a) Structure of the eukaryotic proteasome

Core

proteasome

(4 rings)

(b) Steps of protein degradation in eukaryotic cells

Ubiquitin

Target

protein


71

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